Essential Endocrinology and Diabetes 6°ed 2012 - Richard IG Holt

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Essential Endocrinology and Diabetes

Essential Endocrinology and Diabetes Richard IG Holt Professor in Diabetes and Endocrinology Faculty of Medicine University of Southampton

Neil A Hanley Professor of Medicine Faculty of Medical & Human Sciences University of Manchester

Sixth edition

A John Wiley & Sons, Ltd., Publication

This edition first published 2012 © 2012 by Richard IG Holt and Neil A Hanley Fifth edition © 2007 Richard Holt and Neil Hanley Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form Wiley-Blackwell. Registered office:  John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices:  9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Holt, Richard I. G.   Essential endocrinology and diabetes. – 6th ed. / Richard I.G. Holt, Neil A. Hanley.     p. ; cm. –  (Essentials)   Includes bibliographical references and index.   ISBN-13: 978-1-4443-3004-5 (pbk. : alk. paper)   ISBN-10: 1-4443-3004-7 (pbk. : alk. paper)  1.  Endocrinology–Case studies.  2.  Diabetes–Case studies.  I.  Hanley, Neil A.  II.  Title.  III.  Series: Essentials (Wiley-Blackwell)   [DNLM:  1.  Endocrine Glands–physiology.  2.  Diabetes Mellitus.  3.  Endocrine System Diseases.  4.  Hormones– physiology.  WK 100]   RC648.H65 2012   616.4–dc23 2011024249 A catalogue record for this book is available from the British Library. Set in 10 on 12 pt Adobe Garamond Pro by Toppan Best-set Premedia Limited 1  2012

Contents Preface List of abbreviations How to get the best out of your textbook

PART 1: Foundations of Endocrinology 1 2 3 4

Overview of endocrinology Cell biology and hormone synthesis Molecular basis of hormone action Investigations in endocrinology and diabetes

PART 2: Endocrinology – Biology to Clinical Practice

vii x xii

1 3 14 27 48 63

5 The hypothalamus and pituitary gland 6 The adrenal gland 7 Reproductive endocrinology 8 The thyroid gland 9 Calcium and metabolic bone disorders 10 Pancreatic and gastrointestinal endocrinology and endocrine neoplasia

65 99 127 165 190

PART 3: Diabetes and Obesity

233

11  Overview of diabetes 12 Type 1 diabetes 13 Type 2 diabetes 14 Complications of diabetes 15 Obesity

235 257 285 311 343

Index

213

360

Preface There have been significant advances and developments in the 4 years since we wrote the last edition. Consequently, many areas of the book have required substantial updating and extensive re-writing. Nevertheless, the structure of the book has remained similar to the last edition, which seemed popular around the world. The first part strives to create a knowledgeable reader prepared for the clinical sections. Recognizing that many students now come to medicine from non-scientific backgrounds, we have tried to limit assumptions on prior knowledge. For instance, the concept of negative feedback regulation, covered in Chapter 1, is mandatory for understanding almost all endocrine physiology and is vital for the interpretation of many clinical tests. Similarly, molecular diagnostics has advanced far beyond the historical development of immunoassays. New modalities, such as molecular genetics, mass spectrometry and sophisticated imaging, are already standard practice and it is important that aspiring clinicians, as well as scientists, appreciate their methodology, application and limitations. The second part retains a largely organ-based approach. The introductory basic science in these chapters aims to be concise yet sufficient to understand, diagnose and manage the associated clinical disorders. The chapter on endocrine neoplasia, including hormone-secreting tumours of the gut, has been expanded in recognition of the increasing array of hormones discovered from the pancreas and gastrointestinal tract. In previous editions these hormones have lacked attention. However, many of them are now emerging as key regulators that are exploited in new therapies. For instance, augmentation of glucagon-like peptide 1 signalling is an effective treatment for diabetes. The third part on diabetes and obesity was entirely new in the last edition and these chapters have undergone the greatest change here. Over the last 4 years we have seen significant advances in the treatment of type 2 diabetes such as the new incretinbased therapies and the withdrawal of other treatments due to safety concerns. Clinical algorithms have also changed and these have been updated. The textbook aims to bridge the gap from basic science training, through clinical training, to the knowledge required for the early postgraduate years and specialist training. The text goes beyond core undergraduate medical education. Learning objectives, boxes, and concluding ‘key points’ aim to emphasize the major topics. There is hopefully useful detail for more advanced clinicians who, like the authors, enjoy trying to interpret clinical medicine scientifically, but for whom memory occasionally fails. Although the structure of the book is largely unchanged from the previous edition, readers of the old edition will recognize welcome developments. For the first time, the book is in full colour, which has allowed us to include colour photographs in the relevant chapter. We have introduced recap and cross-reference guides at the beginning of each of the clinical chapters to help the reader find important information in other parts of the book more easily. The case histories that were introduced in the last edition proved to be a success and these have been expanded to provide greater opportunity to put theory into practice. We have brought our clinical and research experiences together to create this book. While it has been a truly collaborative venture and the book is designed to read as a whole, inevitably one of us has taken a lead with each chapter depending on our own interests. As such, NAH was responsible for writing Part 1 and Part 2, while RIGH was responsible for Part 3.

viii  /  Preface

Finally, we must thank a number of people without whom this book would not have come to fruition. We are grateful for the skilled help of Wiley-Blackwell Publishing and remain indebted to our predecessors up to and including the 4th edition, Charles Brook and Nicholas Marshall, for their excellent starting point. We are also grateful to our families without whose support this book would not have been possible and to whom we dedicate this edition. R.I.G. Holt University of Southampton N.A. Hanley University of Manchester

The authors Richard Holt is Professor in Diabetes and Endocrinology at the University of Southampton School of Medicine and Honorary Consultant in Endocrinology at Southampton University Hospitals NHS Trust. His research interests are broadly focused around clinical diabetes with particular interests in diabetes in pregnancy and young adults, and the relation between diabetes and mental illness. He also has a long-standing interest in growth hormone.

Neil Hanley is Professor of Medicine and Wellcome Trust Senior Fellow in Clinical Science at the University of Manchester. He is Honorary Consultant in Endocrinology at the Central Manchester University Hospitals NHS Foundation Trust where he provides tertiary referral endocrine care. His main research interests are human developmental endocrinology and stem cell biology.

Both authors play a keen role in the teaching of undergraduate medical students and doctors. RIGH is a Fellow of the Higher Education Academy. NAH is Director of the Academy for Training & Education at the Manchester Biomedical Research Centre.

Preface  /  ix

Further reading The following major international textbooks make an excellent source of secondary reading: Melmed S, Polonsky KS, Reed Larsen P, Kronenberg HM, eds. Williams Textbook of Endocrinology, 12th edn. Saunders, 2011. Holt RIG, Cockram C, Flyvbjerg A, Goldstein BJ. Textbook of Diabetes, 4th edn. WileyBlackwell, 2010. In addition, the following textbooks cover topics, relevant to some chapters, in greater detail: Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s Essential Immunology, 12th edn. Wiley-Blackwell, 2011. Johnson M. Essential Reproduction, 6th edn. Wiley-Blackwell, 2007. Nelson DL, Cox MM. Lehninger Principles of Biochemistry, 5th edn. W.H. Freeman, 2008.

List of abbreviations 5-HIAA 5-HT αMSH ACTH ADH AFP AGE AGRP AI AII ALS AMH AR APS-1 APS-2 CAH cAMP CBG cGMP CRE CREB CNS CRH CSF CT CVD DAG DEXA DHEA DHT DI EGF EPO ER FFA FGF FIA FISH FSH fT3 fT4

5-hydroxyindoleacetic acid 5-hydroxytryptophan α-melanocyte stimulating hormone adrenocorticotrophic hormone vasopressin/antidiuretic hormone α-fetoprotein advanced glycation end-product Agouti-related protein angiotensin I angiotensin II acid labile subunit anti-Müllerian hormone androgen receptor type 1 autoimmune polyglandular syndrome type 2 autoimmune polyglandular syndrome congenital adrenal hyperplasia cyclic adenosine monophosphate cortisol binding globulin guanosine monophosphate cAMP response element cAMP response element-binding protein central nervous system corticotrophin-releasing hormone cerebrospinal fluid computed tomography cardiovascular disease diacylglycerol dual energy X-ray absorptiometry dehydroepiandrosterone 5α-dihydrotestosterone diabetes insipidus epidermal growth factor erythropoietin oestrogen receptor free fatty acid fibroblast growth factor fluoroimmunoassay fluorescence in situ hybridization follicle-stimulating hormone free tri-iodothyronine free thyroxine

GC GDM GFR GH GHR GHRH GI GIP

gas chromatography gestational diabetes glomerular filtration rate growth hormone (somatotrophin) GH receptor growth hormone-releasing hormone glycaemic index glucose-dependent insulinotrophic peptide (gastric inhibitory peptide) GLUT glucose transporter GnRH gonadotrophin-releasing hormone GPCR guanine-protein coupled receptor GR glucocorticoid receptor Grb2 type 2 growth factor receptor-bound protein hCG human chorionic gonadotrophin hMG human menopausal gonadotrophin HMGCoA hydroxymethylglutaryl coenzyme A HNF hepatocyte nuclear factor HPLC high performance liquid chromatography HRE hormone response element HRT hormone replacement therapy ICSI intracytoplasmic sperm injection IDDM insulin-dependent diabetes mellitus IFG impaired fasting glycaemia IFMA immunofluorometric assay IGF insulin-like growth factor IGFBP IGF-binding protein IGT impaired glucose tolerance IP inositol phosphate IPF insulin promoter factor IR insulin receptor IRMA intraretinal microvascular abnormalities (Chapter 14) IRMA immunoradiometric assay (Chapter 4) IRS insulin receptor substrate IVF in vitro fertilization JAK Janus-associated kinase LDL low-density lipoprotein LH luteinizing hormone MAO monoamine oxidase MAPK mitogen-activated protein kinase

List of abbreviations  /  xi

MEN MIS MODY MR MRI MS MSH NEFA NICTH NIDDM NPY NVD NVE OGTT PCOS PCR PDE PGE2 PI PIT1 PKA PKC PLC PNMT POMC PPAR PRL PTH PTHrP PTU

multiple endocrine neoplasia Müllerian inhibiting substance maturity-onset diabetes of the young mineralocorticoid receptor magnetic resonance imaging mass spectrometry melanocyte-stimulating hormone non-esterified fatty acid non-islet cell tumour hypoglycaemia non–insulin-dependent diabetes mellitus neuropeptide Y new vessels at the disc new vessels elsewhere oral glucose tolerance test polycystic ovarian syndrome polymerase chain reaction phosphodiesterase prostaglandin E2 phosphatidylinositol pituitary-specific transcription factor 1 protein kinase A protein kinase C phospholipase C phenylethanolamine N-methyl transferase pro-opiomelanocortin peroxisome proliferator-activated receptor prolactin parathyroid hormone parathyroid hormone-related peptide propylthiouracil

RANK RER RIA rT3 RXR SERM SHBG SIADH SoS SRE SS StAR STAT T1DM T2DM t1/2 T3 T4 TGFβ TK TPO TR TRE TRH TSH UFC V VEGF VIP

receptor activator of nuclear factor-kappa B rough endoplasmic reticulum radioimmunoassay reverse tri-iodothyronine retinoid X receptor selective ER modulator sex hormone-binding globulin syndrome of inappropriate antidiuretic hormone son of sevenless protein serum response element somatostatin steroid acute regulatory protein signal transduction and activation of transcription protein type 1 diabetes type 2 diabetes half-life tri-iodothyronine thyroxine transforming growth factor β tyrosine kinase thyroid peroxidase thyroid hormone receptor thyroid hormone response element thyrotrophin-releasing hormone thyroid-stimulating hormone urinary free cortisol vasopressin/antidiuretic hormone (previously also known as arginine vasopressin) vascular endothelial growth factor vasoactive intestinal peptide

How to get the best out of your textbook Welcome to the new edition of Essential Endocrinology and Diabetes. Over the next few pages you will be shown how to make the most of the learning features included in the textbook. The anytime, anywhere textbook  For the first time, your textbook comes with free access to a Wiley Desktop Edition – a digital, interactive version of this textbook which you own as soon as you download it. Your Wiley Desktop Edition allows you to: Search: Save time by finding terms and topics instantly in your book, your notes, even your whole library (once you’ve downloaded more textbooks) Note and Highlight: Colour code highlights and make digital notes right in the text so you can find them quickly and easily Organize: Keep books, notes and class materials organized in folders inside the application Share: Exchange notes and highlights with friends, classmates and study groups Upgrade: Your textbook can be transferred when you need to change or upgrade computers To access your Wiley Desktop Edition: •  Find the redemption code on the inside front cover of this book and carefully scratch away the top coating of the label. • Visit www.vitalsource.com/software/ bookshelf/downloads to download the Bookshelf application to your computer, laptop or mobile device • Open the Bookshelf application on your computer and register for an account. • Follow the registration process and enter your redemption code to download your digital book.

How to get the best out of your textbook  / xiii xiii Chapter 7:  /   / 

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Features contained within your textbook

3

CHAPTER 1

Overview of endocrinology

5

■ Classification of hormones

8

■ Organization and control of endocrine organs

9 13

■ Key points

13

VI

■ Endocrine disorders

Learning objectives

■ To be able to define endocrinology

■ To understand what endocrinology is as a basic science and

a clinical specialty

■ To appreciate the history of endocrinology

RE

■ To understand the classification of hormones into peptides,

steroids and amino acid derivatives

■ To understand the principle of feedback mechanisms that

regulate hormone production

This chapter details some of the history to endocrinology and d diabetes, and introduces basic principles that underpin the subsequent chapters

Complications of diabetes

D

4

■ The role of hormones

Key topics

SE

■ A brief history of endocrinology and diabetes

■ Microvascular complications ■ Macrovascular disease ■ Cancer

312 330 334

■ Psychological complications

334

■ How diabetes care can reduce complications

335

■ Diabetes and pregnancy

336

■ Social aspects of diabetes

339

■ Key points

340

■ Answers to case histories

340

VI

Key topics

SE

D

311

CHAPTER 14

 Every chapter has its own chapter-opening page that offers a list of key topics and learning objectives pertaining to the chapter Throughout your textbook you will find this icon which points you to cases with self-test questions. You will find the answers at the end of each chapter.

Learning objectives

RE

■ To discuss the causes of microvascular and macrovascular

complications

■ To understand the importance of screening for complications onss ■ To understand the strategies to prevent and treat

complications

■ To discuss diabetes in pregnancy

Essential Endocrinology and Diabetes, Sixth Edition. Richard IG Holt, Neil A Hanley. © 2012 Richard IG Holt and Neil A Hanley. Publlished 2012 by Blackwell Publishing Ltd.

■ To understand the psychosocial aspects of diabetes

This chapter discusses the microvascular and macrovascular complications of diabetes, diabetes in pregnancy and psychosocial aspects of diabetes

Essential Endocrinology and Diabetes, Sixth Edition. Richard IG Holt, Neil A Hanley. © 2012 Richard IG Holt and Neil A Hanley. Publlished 2012 by Blackwell Publishing Ltd.

You will also find a helpful list of key points at the end of each chapter which give a quick summary of important principles.

xiv  /  How to get the best out of your textbook

102 / Chapter 6: The adrenal gland

Figure 6.2 Section through the adrenal cortex. (a) The blood vessels run from outer capsule to medullary venule. (b) A zona fasciculata cell with large lipid droplets, extensive smooth endoplasmic reticulum and mitochondria.

 Your textbook is full of useful photographs, illustrations and tables. The Desktop Edition version of your textbook will allow you to copy and paste any photograph or illustration into assignments, presentations and your own notes. 

Chapter 6: The adrenal gland / 103

(a) Capsule

(b) Smooth endoplasmic reticulum

Zona glomerulosa

17,20-lyase (CYP17A1)

17α-hydroxylase (CYP17A1)

Sulphotransferase (SULT2A1)

Cholesterol Cholesterol side chain cleavage (CYP11A1)

Capillary

Zona fasciculata Lipid droplets Mitochondria

21-hydroxylase (CYP21A2)

Zona reticularis

Pregnenolone

17a-hydroxypregnenolone

Progesterone

17a-hydroxyprogesterone

3β-hydroxysteroid dehydrogenase (HSD3B2)

11β-hydroxylase (CYP11B2)

Venule

DHEA sulphate (DHEAS)

Androstenedione

21-hydroxylase (CYP21A2) Deoxy11-deoxycortisol corticosterone 11β-hydroxylase (CYP11B1) Corticosterone

Medulla

Dehydroepi(DHEA) androsterone

Cortisol

18-hydroxylase (CYP11B2) 18-hydroxycorticosterone Aldosterone synthase (CYP11B2)

Box 6.2 Zones of the adult adrenal cortex and their steroid hormone secretion • Zona glomerulosa, nests of closely packed small cells creating the thinnest, outermost layer; secretes aldosterone • Zona fasciculata, larger cells in columns making up ∼three-quarters of the cortex; secretes cortisol and some sex steroid precursors • Zona reticularis, ‘net-like’ arrangements of innermost cells, formed at 6–8 years to herald a poorly understood change called ‘adrenarche’; secretes sex steroid precursors and some cortisol

cortisol measurement; an important clinical issue. Like thyroid hormones, it is only the free component that enters target cells. The free component also passes into urine – ‘urinary free cortisol’ (UFC) – where its collection and assay over 24 h has been

used as a test of glucocorticoid excess (UFC is ∼1% of cortisol production by the adrenal glands). Measuring salivary cortisol similarly avoids issues with fluctuating serum CBG. Regulation of cortisol biosynthesis and secretion – the hypothalamic–pituitary– adrenal axis The major regulation of the adrenal cortex comes from the anterior pituitary, via the production and circulation of adrenocorticotrophic hormone (ACTH), a cleavage product of the proopiomelanocortin (POMC) gene (review Chapter 5). In turn, ACTH is regulated by corticotrophinreleasing hormone (CRH) from the hypothalamus (Figure 6.4). By binding to cell-surface receptors and activating cAMP second messenger pathways, ACTH increases flux through the pathway from cholesterol to cortisol, particularly at the ratelimiting step catalyzed by CYP11A1. This happens acutely such that a rise in ACTH increases cortisol levels within 5 min. Cortisol provides feedback to the anterior pituitary and hypothalamus to

Aldosterone

Figure 6.3 Biosynthesis of adrenocortical steroid hormones. HSD3B activity in the adrenal cortex arises from the type 2 isoform. The three steps from deoxycorticosterone to aldosterone are catalyzed by the same enzyme,252 / Chapter 11: Overview of diabetes CYP11B2 (also see Figure 2.6).

• Transport of cholesterol into the mitochondrion by the steroid acute regulatory (StAR) protein • The rate-limiting removal of the cholesterol side chain by CYP11A1 • Shuttling intermediaries between mitochondria and endoplasmic reticulum for further enzymatic modification • Action of two key enzymes at branch points: ° CYP17A1 prevents the biosynthesis of aldosterone and commits a steroid to

cortisol or sex steroid precursor. Hence, CYP17A1 is absent from the zona glomerulosa

° Early action of HSD3B2 steers steroid

precursors away from the sex steroid precursors towards aldosterone or cortisol • The presence and activity of CYP11B2 in the zona glomerulosa permitting aldosterone synthesis • The presence and activity of CYP11B1 in the fasciculata (and less so the reticularis) zone allowing cortisol production

Cross-reference

Chapter 11: Overview of diabetes / 253

Table 11.6 Insulin actions on carbohydrate metabolism

Box 6.3 Adrenocortical steroidogenesis can be summarized into a few key steps

Action

Mechanism

Increases glucose uptake into cells

Translocation of glucose transporter (GLUT)-4 to the cell surface

Increases glycogen synthesis

Activates glycogen synthase by dephosphorylation

Inhibits glycogen breakdown

Inactivates glycogen phosphorylase and its activating kinase by dephosphorylation

Inhibits gluconeogenesis

Dephosphorylation of pyruvate kinase and 2,6-biphosphate kinase

Increases glycolysis

Dephosphorylation of pyruvate kinase and 2,6-biphosphate kinase

Converts pyruvate to acetyl CoA

Activates the intramitochondrial enzyme complex pyruvate dehydrogenase

phosphorylation and dephosphorylation of the enzymes controlling glycogenolysis and glycogen synthesis (Figure 11.12). The rate-limiting enzymes are the catabolic enzyme glycogen phosphorylase and the anabolic enzyme glycogen synthase. Insulin increases glycogen synthesis by activating glycogen synthase, while inhibiting glycogenolysis by dephosphorylating glycogen phosphorylase kinase. Glycolysis is stimulated and gluconeogenesis inhibited by dephosphorylation of pyruvate kinase (PK) and 2,6-biphosphate kinase. Insulin also enhances the irreversible conversion of pyruvate to acetyl CoA by activation of the intramitochondrial enzyme complex pyruvate dehydrogenase. Acetyl CoA may then be directly oxidized via the tricarboxylic acid (Krebs’) cycle, or used for fatty acid synthesis (Figure 11.13). Lipid metabolism Insulin increases the rate of lipogenesis in several ways in adipose tissue and liver, and controls the formation and storage of triglyceride. The critical step in lipogenesis is the activation of the insulin-sensitive lipoprotein lipase in the capillaries. Fatty acids are then released from circulating chylomicrons or very low-density lipoproteins and taken up into the adipose tissue. Fatty acid synthesis is increased by activation and increased phosphorylation of acetyl CoA carboxylase, while fat oxidation is suppressed by inhibition of carnitine acyltransferase (Table 11.7). Lipogenesis is also facilitated by glucose uptake, because its metabo-

Glucose

lism by the pentose phosphate pathway provides nicotinamide adenine dinucleotide phosphate (NADPH), which is needed for fatty acid synthesis. Triglyceride synthesis is stimulated by esterification of glycerol phosphate, while triglyceride breakdown is suppressed by dephosphorylation of hormone-sensitive lipase. Cholesterol synthesis is increased by activation and dephosphorylation of hydroxymethylglutaryl co-enzyme A (HMGCoA) reductase, while cholesterol ester breakdown appears to be inhibited by dephosphorylation of cholesterol esterase. Phospholipid metabolism is also influenced by insulin. Protein metabolism Insulin stimulates the uptake of amino acid into cells and promotes protein synthesis in a range of tissues. There are effects on the transcription of specific mRNA, as well as their translation into proteins on the ribosomes (review Chapter 2 and Figure 2.2). Examples of enhanced mRNA transcription include glucokinase and fatty acid synthase. By contrast, insulin decreases mRNA encoding liver enzymes such as carbamoyl phosphate synthetase, which is a key enzyme in the urea cycle. However, the major action of insulin on protein metabolism is to inhibit the breakdown of proteins (Figure 11.14). In this way it acts synergistically with GH and insulin-like growth factor I (IGF-I) to increase protein anabolism.

Glucose-6- P

Sorbitol route

Fructose

Glucose-1- P

Glycogen

UDP-Glucose Glycolyticgluconeogenic pathways α-Glycero- P Lactate

Pyruvate

NADPH

Tricarboxylic acid cycle Protein degradation

Pentose phosphate pathway

NADP+

Glycoprotein Mucopolysaccharide

Ribose-5- P

Nucleotides RNA and DNA

α-Glycero- P Lipid synthesis

Acetyl CoA

ATP Amino acids

Glucuronatexylulose pathway

Urea cycle

NH3

Figure 11.12 Inter-relationships among alternative routes of glucose metabolism. The central role of glucose in carbohydrate, fat and protein metabolism is summarized. The principal metabolic pathways are shown enclosed in boxes in order to simplify the diagram; some key intermediates and products of metabolic interconversions are shown. The

Triglycerides

Urea

reversibility of certain reaction sequences implied by double-headed arrows is not necessarily intended to suggest that the same enzymes are involved in both the forward and reverse reactions. The principal reversible pathways that are activated during fasting are marked with heavy arrows.

Table 11.7 Insulin actions on fatty acid metabolism Action

Mechanism

Releases fatty acids from circulating chylomicrons or very low-density lipoproteins

Activates lipoprotein lipase

Increases fatty acid synthesis

Activates acetyl CoA carboxylase

Suppresses fatty acid oxidation

Inhibits carnitine acyltransferase

Increases triglyceride synthesis

Stimulates esterification of glycerol phosphate

Inhibits triglyceride breakdown

Dephosphorylates hormone-sensitive lipase

Increases cholesterol synthesis

Activates and dephosphorylates HMGCoA reductase

Inhibits cholesterol ester breakdown

Dephosphorylates cholesterol esterase

The development of the parathyroid and parafollicular C-cells is described alongside the thyroid in Chapter 8 Tumours of the parathyroid glands are an important component of multiple endocrine neoplasia, covered in Chapter 10 Other hormones such as cortisol (see Chapter 6) and sex hormones (see Chapter 7) affect mineralization of the bones

 At the beginning of some chapters you will also find cross-references which make it easy to locate related information quickly and efficiently.

We hope you enjoy using your new textbook. Good luck with your studies!

Part 1 Foundations of Endocrinology

3  

CHAPTER 1

Overview of endocrinology Key topics ■ A brief history of endocrinology and diabetes

4

■ The role of hormones

5

■ Classification of hormones

8

■ Organization and control of endocrine organs

9

■ Endocrine disorders

13

■ Key points

13

Learning objectives ■ To be able to define endocrinology ■ To understand what endocrinology is as a basic science and

a clinical specialty ■ To appreciate the history of endocrinology ■ To understand the classification of hormones into peptides,

steroids and amino acid derivatives ■ To understand the principle of feedback mechanisms that

regulate hormone production This chapter details some of the history to endocrinology and diabetes, and introduces basic principles that underpin the subsequent chapters

Essential Endocrinology and Diabetes, Sixth Edition. Richard IG Holt, Neil A Hanley. © 2012 Richard IG Holt and Neil A Hanley. Publlished 2012 by Blackwell Publishing Ltd.

4  /  Chapter 1: Overview of endocrinology

An organism comprised of a single or a few cells analyzes and responds to its external environment with relative ease. No cell is more than a short diffusion distance from the outside world or its neighbours, allowing a constancy of internal environment (‘homeostasis’). This simplicity has been lost with the evolution of more complex, larger, multicellular organisms. Simple diffusion has become inadequate in larger animal species where functions localize to specific organs. In humans, there are ∼1014 cells of 200 or more different types. With this compartmentalized division of purpose comes the need for effective communication to disseminate information throughout the whole organism – only a few cells face the outside world, yet all respond to it. Two communication systems facilitate this: the endocrine and nervous systems (Box 1.1). The specialized ductless glands and tissues of the endocrine system release chemical messengers – hormones – into the extracellular space, from where they enter the bloodstream. It is this blood-borne transit that defines endocrinology; however, the principles are similar for hormone action on a neighbouring cell (‘paracrinology’) or, indeed, itself (‘auto- or intra-crinology’) (Figure 1.1). The nervous and endocrine systems interact. Endocrine glands are under both nervous and hormonal control, while the central nervous system is affected by multiple hormonal stimuli – features reflected by the composite science of neuroendocrinology (Figure 1.1).

A brief history of endocrinology and diabetes The term ‘hormone’, derived from the Greek word ‘hormaein’ meaning ‘to arouse’ or ‘to excite’, was first used in 1905 by Sir Ernest Starling in his Croonian Lecture to the Royal College of Physicians;

(a) Endocrine

(b) Autocrine (A) and Paracrine (P) Target cell

Hormone

Target cell

A

P

Local hormone

Blood vessel

(c) Neuroendocrine Nerve cell

(d) Neurotransmitter Neurone

Axon

Axon terminal

Neurone Neurotransmitter

Blood vessel Target cell

Box 1.1  Functions of the endocrine and nervous systems, the two main communication systems •  To monitor internal and external environments • To allow appropriate adaptive changes • To communicate via chemical messengers

}

maintain homeostasis

Figure 1.1  Cells that secrete regulatory substances to communicate with their target cells and organs. (a) Endocrine. Cells secrete hormone into the blood vessel, where it is carried, potentially over large distances, to its target cell. (b) Autocrine (A): hormones such as insulin-like growth factors can act on the cell that produces them, representing autocrine control. Paracrine (P): cells secrete hormone that acts on nearby cells (e.g. glucagon and somatostatin act on adjacent β-cells within the pancreatic islet to influence insulin secretion). (c) Stimulated neuroendocrine cells secrete hormone (e.g. the hypothalamic hormones that regulate the anterior pituitary) from axonic terminals into the bloodstream. (d) Neurotransmitter cells secrete substances from axonic terminals to activate adjacent neurones.

Chapter 1: Overview of endocrinology  /  5

however, the specialty is built on foundations that are far older. Aristotle described the pituitary, while the associated condition, gigantism, due to excess growth hormone (GH), was referred to in the Old Testament, two millennia or so before the 19th century recognition of the gland’s anterior and posterior components by Rathke, and Pierre Marie’s connection of GH-secreting pituitary tumours to acromegaly. Diabetes was recognized by the ancient Egyptians. Areteus later described the disorder in the second century ad as ‘a melting down of flesh and limbs into urine’ – diabetes comes from the Greek word meaning siphon. The pancreas was only implicated relatively recently when Minkowski realized in 1889 that the organ’s removal in dogs mimicked diabetes in humans. The roots of reproductive endocrinology are equally long. The Bible refers to eunuchs and Hippocrates recognized that mumps could result in sterility. Oophorectomy in sows and camels was used to increase strength and growth in ancient Egypt. The association with technology is also longstanding. For instance, it took the microscope in the 17th century for Leeuwenhoek to visualize spermatozoa and later, in the 19th century, for the mammalian ovum to be discovered in the Graafian follicle. During the last 500 years, other endocrine organs and axes have been identified and characterized. In 1564, Bartolommeo Eustacio noted the presence of the adrenal glands. Almost 300 years later (1855), Thomas Addison, one of the forefathers of clinical endocrinology, described the consequences of their inadequacy. Catecholamines were identified at the turn of the 19th century, in parallel with Oliver and Schaffer’s discovery that these adrenomedullary substances raise blood pressure. This followed shortly after the clinical features of myxoedema were linked to the thyroid gland, when, in 1891, physicians in Newcastle treated hypothyroidism with sheep thyroid extract. This was an important landmark, but long after the ancient Chinese recognized that seaweed, as a source of iodine, held valuable properties in treating ‘goitre’, swelling of the thyroid gland. Early clinical endocrinology and diabetes tended to recognize and describe the features of the endo-

crine syndromes. Since then, our understanding has advanced through: • Successful quantification of circulating hormones • Pathophysiological identification of endocrine dysfunction • Molecular genetic diagnoses • Molecular unravelling of complex hormone action. Some of the landmarks from the last 100 years are shown in Box 1.2, and those researchers who have been awarded the Nobel Prize for Medicine, Physiology or Chemistry for discoveries that have advanced endocrinology and diabetes are listed in Table 1.1. Traditionally, endocrinology has centred on specialized hormone-secreting organs (Figure 1.2), largely built on the ‘endocrine postulates’ of Edward Doisy (Box 1.3). While the focus of this textbook remains with these organs, many tissues display appreciable degrees of hormone biosynthesis, and, equally relevant, modulate hormone action. All aspects are important for a complete appreciation of endocrinology and its significance.

The role of hormones Hormones are synthesized by specialized cells (Table 1.2), which may exist as distinct endocrine glands or be located as single cells within other organs, such as the gastrointestinal tract. The chapters in Part 2 are largely organized on this anatomical basis. Endocrinology is defined by the secretion of hormones into the bloodstream; however, autocrine or paracrine actions are also important, often modulating the hormone-secreting cell type. Hormones act by binding to specific receptors, either on the surface of or inside the target cell, to initiate a cascade of intracellular reactions, which frequently amplifies the original stimulus and generates a final response. These responses are altered in hormone deficiency and excess: for instance, GH deficiency leads to short stature in children, while excess causes over-growth (either gigantism or acromegaly; Chapter 5).

6  /  Chapter 1: Overview of endocrinology

Box 1.2  Some landmarks in endocrinology over the last 100 years or so 1905 1909 1914 1921 Early 1930s 1935–40 1935–40 1940s 1952 1955 1956 1950s

1955 1957 1966 1969 1969–71 1973 1981–2 1994 1994 1999 1999 2000

First use of the term ‘hormone’ by Starling in the Croonian Lecture at the Royal College of Physicians Cushing removed part of the pituitary and saw improvement in acromegaly Kendall isolated an iodine-containing substance from the thyroid Banting and Best extracted insulin from islet cells of dog pancreas and used it to lower blood sugar Pitt-Rivers and Harrington determined the structure of the thyroid hormone, thyroxine Crystallization of testosterone Identification of oestrogen and progesterone Harris recognized the relationship between the hypothalamus and anterior pituitary in the ‘portal-vessel chemotransmitter hypothesis’ Gross and Pitt-Rivers identified tri-iodothyronine in human serum The Schally and Guillemin laboratories showed that extracts of hypothalamus stimulated adrenocorticotrophic hormone (ACTH) release Doniach, Roitt and Campbell associated antithyroid antibodies with some forms of hypothyroidism – the first description of an autoimmune phenomenon Adams and Purves identified thyroid stimulatory auto-antibodies Gonadectomy and transplantation experiments by Jost led to the discovery of the role for testosterone in rabbit sexual development Sanger reported the primary structure of insulin Growth hormone was used to treat short stature in patients First transplant of human pancreas to treat type 1 diabetes by Kelly, Lillehei, Goetz and Merkel at the University of Minnesota Hodgkin reported the three-dimensional crystallographic structure of insulin Discovery of thyrotrophin-releasing hormone (TRH) and gonadotrophin-releasing hormone (GnRH) by Schally’s and Guillemin’s groups Discovery of somatostatin by the group of Guillemin Discovery of corticotrophin-releasing hormone (CRH) and growth hormonereleasing hormone (GHRH) by Vale Identification of leptin by Friedman and colleagues First transplantation of pancreatic islets to treat type 1 diabetes by Pipeleers and colleagues in Belgium Discovery of ghrelin by Kangawa and colleagues Sequencing of the human genome – publication of the DNA code for chromosome 22 Advanced islet transplantation using modified immunosuppression by Shapiro and colleagues to treat type 1 diabetes

Thyroid hormone acts on many, if not all, of the 200 plus cell types in the body. The basal metabolic rate increases if it is present in excess and declines if there is a deficiency (see Chapter 8). Similarly, insulin acts on most tissues, implying its receptors are widespread. Its importance is also underlined by

its role in the survival and growth of many cell types in laboratory culture. In contrast, other hormones may act only on one tissue. Thyroid-stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH) and the gonadotrophins are secreted by the anterior pituitary and have specific target tissues

Chapter 1: Overview of endocrinology  /  7

Table 1.1  Nobel prize winners for discoveries relevant to endocrinology and diabetes Year

Prize winner(s)

For work on . . . 

1909

Emil Theodor Kocher

Physiology, pathology and surgery of the thyroid gland

1923

Frederick Grant Banting and John James Richard Macleod

Discovery of insulin

1928

Adolf Otto Reinhold Windhaus

Constitution of the sterols and their connection with the vitamins

1939

Adolf Friedrich and Johann Butenandt

Sex hormones

1943

George de Hevesy

Use of isotopes as tracers in the study of chemical processes

1946

James Batcheller Summer, John Howard Northrop and Wendell Meredith Stanley

Discovery that enzymes can be crystallized and prepared in a pure form

1947

Carl Ferdinand Cori, Getty Theresa Cori (neé Radnitz) and Bernardo Alberto Houssay

Discovery of the course of the catalytic conversion of glycogen

1950

Edwin Calvin Kendall, Tadeus Reichstein and Philip Showalter Hench

Discoveries relating to the hormones of the adrenal cortex, their structure and biological effects

1955

Vincent du Vigneaud

Biochemically important sulphur compounds, especially for the first synthesis of a polypeptide hormone

1958

Frederick Sanger

Structures of proteins, especially that of insulin

1964

Konrad Bloch and Feodor Lynen

Discoveries concerning the mechanism and regulation of cholesterol and fatty acid metabolism

1966

Charles Brenton Huggins

Discoveries concerning hormonal treatment of prostatic cancer

1969

Derek HR Barton and Odd Hassel

Development of the concept of conformation and its application in chemistry

1970

Bernard Katz, Ulf von Euler and Julius Axelrod

Discoveries concerning the humoral transmitters in the nerve terminals and the mechanism for their storage, release and inactivation

1971

Earl W Sutherland Jr

Discoveries concerning the mechanisms of the action of hormones

1977

Roger Guillemin, Andrew V Schally and Rosalyn Yalow

Discoveries concerning peptide hormones in the production in the brain and the development of radioimmunoassay from peptide hormones

1979

Allan M Cormack and Godfrey N Hounsfield

Development of computer-assisted tomography (Continued )

8  /  Chapter 1: Overview of endocrinology

Table 1.1  (Continued ) Year

Prize winner(s)

For work on . . . 

1982

Sune K Bergström, Bengt I Samuelson and John R Vane

Discoveries concerning prostaglandins and related biologically active substances

1985

Michael S Brown and Joseph L Goldstein

Discoveries concerning the regulation of cholesterol metabolism

1986

Stanley Cohen and Rita Levi-Montalcini

Discoveries of growth factors

1992

Edmond H Fischer and Edwin G Krebs

Discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism

1994

Alfred G Gilman and Martin Rodbell

Discovery of G-proteins and the role of these proteins in signal transduction in cells

2003

Peter Agre and Roderick MacKinnon

Discovery of water channels, and the structural and mechanistic studies of ion channels

2003

Paul Lauterbur and Sir Peter Mansfield

Discoveries concerning magnetic resonance imaging

2010

Robert G Edwards

Development of in vitro fertilization

– the thyroid gland, the adrenal cortex and the gonads, respectively (Table 1.2).

Classification of hormones There are three major groups of hormones according to their biochemistry (Box 1.4). Peptide or protein hormones are synthesized like any other cellular protein. Amino acid-derived and steroid hormones originate from a cascade of biochemical reactions catalyzed by a series of intracellular enzymes. Peptide hormones The majority of hormones are peptides and range in size from very small, only three amino acids [thyrotrophin-releasing hormone (TRH)], to small proteins of over 200 amino acids, such as TSH or luteinizing hormone (LH). Some peptide hormones are secreted directly, but most are stored in granules, the release from which is commonly controlled by another hormone, as part of a cascade, or by innervation.

Some peptide hormones have complex tertiary structures or are comprised of more than one peptide chain. Oxytocin and vasopressin, the two posterior pituitary hormones, have ring structures linked by disulphide bridges. Despite being remarkably similar in structure, they have very different physiological roles (Figure 1.3). Insulin consists of α- and β-chains linked by disulphide bonds. Like several hormones, it is synthesized as an inactive precursor that requires modification prior to release and activity. To some extent this regulation protects the synthesizing cell from being overwhelmed by its own hormone action. The gonadotrophins, folliclestimulating hormone (FSH) and LH, TSH and human chorionic gonadotrophin (hCG) also have two chains. However, these α- and β-subunits are synthesized quite separately, from separate genes. The α-subunit is common; the distinctive β-subunit of each confers biological specificity. Amino acid derivatives These hormones are small water-soluble compounds. Melatonin is derived from tryptophan, whereas tyrosine derivatives include thyroid hor-

Chapter 1: Overview of endocrinology  /  9

Pituitary gland: anterior and posterior Thyroid gland

Two adrenal glands: cortex and medulla Duodenum

Hypothalamus and median eminence Four parathyroid glands

Stomach Kidney Pancreas: Islets of Langerhans Two ovaries (o)

Two testes (o)

Figure 1.2  The sites of the principal endocrine glands. The stomach, kidneys and duodenum are also shown. Not shown, scattered cells within the gastrointestinal tract secrete hormones.

Box 1.3  The ‘Endocrine Postulates’: Edward Doisy, St Louis University School of Medicine, USA, 1936 • The gland must secrete something (an ‘internal secretion’) • Methods of detecting the secretion must be available • Purified hormone must be obtained from gland extracts • The pure hormone must be isolated, its structure determined and synthesized To this could be added: • The hormone must act on specific target cells via a receptor such that excess or deficiency results in a specific phenotype

stituent of the cell membrane. Produced by the adrenal cortex, gonad and placenta, steroid hormones are insoluble in water and largely circulate bound to plasma proteins.

Organization and control of endocrine organs

mones, catecholamines, and dopamine, which regulates prolactin secretion in the anterior pituitary. The catecholamines from the adrenal medulla, epinephrine (adrenaline) and norepinephrine (noradrenaline), are also sympathetic neurotransmitters, emphasizing the close relationship between the nervous and endocrine systems (see Figure 1.2). Like peptide hormones, they are stored in granules prior to release.

The synthesis and release of hormones is regulated by control systems, similar to those used in engineering. These mechanisms ensure that hormone signals can be limited in amplitude and duration. Central to the regulation of many endocrine organs is the anterior pituitary gland, which, in turn, is controlled by a number of hormones and factors released from specialized hypothalamic neurones (see Chapter 5). Thus, major endocrine axes comprise the hypothalamus, anterior pituitary and end organ, such as the adrenal cortex, thyroid, testis or ovary. An understanding of these control mechanisms is crucial for appreciating both regulation of many endocrine systems and their clinical investigation.

Steroid hormones

Simple control

Steroid hormones are lipid-soluble molecules derived from cholesterol, which is itself a basic con-

An elementary control system is one in which the signal itself is limited, either in magnitude or

10  /  Chapter 1: Overview of endocrinology

Table 1.2  The endocrine organs and their hormones* Gland

Hormone

Hypothalamus/ median eminence

Releasing and inhibiting hormones:

Molecular characteristics

  Thyrotrophin-releasing hormone (TRH)

Peptide

  Somatostatin (SS; inhibits GH))

Peptide

  Gonadotrophin-releasing hormone (GnRH)

Peptide

  Corticotrophin-releasing hormone (CRH)

Peptide

  Growth hormone-releasing hormone (GHRH)

Peptide

  Dopamine (inhibits prolactin)

Tyrosine derivative

Thyrotrophin or thyroid-stimulating hormone (TSH)

Glycoprotein

Luteinizing hormone (LH)

Glycoprotein

Follicle-stimulating hormone (FSH)

Glycoprotein

Growth hormone (GH) (also called somatotrophin)

Protein

Prolactin (PRL)

Protein

Adrenocorticotrophic hormone (ACTH)

Peptide

Vasopressin [also called antidiuretic hormone (ADH)]

Peptide

Oxytocin

Peptide

Thyroxine (T4) and tri-iodothyronine (T3)

Tyrosine derivatives

Calcitonin

Peptide

Parathyroid

Parathyroid hormone (PTH)

Peptide

Adrenal cortex

Aldosterone

Steroid

Cortisol

Steroid

Androstenedione

Steroid

Dehydroepiandrosterone (DHEA)

Steroid

Epinephrine (also called adrenaline)

Tyrosine derivative

Norepinephrine (also called noradrenaline)

Tyrosine derivative

Gastrin

Peptide

Insulin

Protein

Glucagon

Protein

Somatostatin (SS)

Protein

Secretin

Protein

Cholecystokinin

Protein

Anterior pituitary

Posterior pituitary

Thyroid

Adrenal medulla

Stomach

†

Pancreas (islets of Langerhans)†

Duodenum and jejunum†

Chapter 1: Overview of endocrinology  /  11

Table 1.2  (Continued ) Gland

Hormone

Molecular characteristics

Liver

Insulin-like growth factor I (IGF-I)

Protein

Ovary

Oestrogens

Steroid

Progesterone

Steroid

Testosterone

Steroid

Testis

*  The distinction between peptide and protein is somewhat arbitrary. Shorter than 50 amino acids is termed a peptide in this table. †   The list is not exhaustive for the gastrointestinal tract and the pancreas (see Chapter 11).

Vasopressin

regulates water excretion 3

8

Cys – Tyr – Phe – Gln – Asn – Cys – Pro – Arg – Gly (NH2) S

Oxytocin

S

Figure 1.3  The structures of vasopressin and oxytocin are remarkably similar, yet the physiological effects of the two hormones differ profoundly.

uterine contraction 3

8

Cys – Tyr – IIe – Gln – Asn – Cys – Pro – Leu – Gly (NH2) S

S

Box 1.4  Major hormone groups • Peptides and proteins • Amino acid derivatives • Steroids

duration, so as to induce only a transient response. Certain neural impulses are of this type. Refinement allows discrimination of a positive signal from background ‘noise’ to ensure that the target cell cannot or does not respond below a certain threshold level. An example is the pulsatile release of gonadotrophin-releasing hormone (GnRH) from the hypothalamus. Negative feedback Negative feedback is the commonest form of regulation used by many biological systems. For example,

in enzymology, the product frequently inhibits further progress of the catalyzed reaction. In endocrinology, a hormone may act on its target cell to stimulate a response (often secretion of another hormone) that then inhibits production of the first hormone (Figure 1.4a). Hormone secretion may also be regulated by metabolic processes. For instance, the pancreatic β-cell makes insulin in response to high ambient glucose. The effect is to lower glucose, which, in turn, inhibits further insulin production. The hypothalamic–anterior pituitary–end organ axes are a more complex extension of this model. The hypothalamic hormone [e.g. corticotrophinreleasing hormone (CRH)] stimulates release of anterior pituitary hormone (e.g. ACTH) to increase peripheral hormone production (e.g. cortisol), which then feeds back on the anterior pituitary and hypothalamus to reduce the original secretions. Figure 1.4b illustrates the anterior pituitary and endorgan components of this model.

12  /  Chapter 1: Overview of endocrinology

(a)

(b) Endocrine organ

+

Endocrine organ

–

Hormone

Response

–

Hormone 1

Hormone 2

Target

Target

tissue

endocrine

Target tissue

organ

Response

Figure 1.4  Two models of feedback regulating hormone synthesis. (a) The endocrine organ releases a hormone, which acts on the target tissue to stimulate a response. The response usually feeds back to inhibit ( – ) the endocrine organ to decrease further supply of the hormone. Occasionally, the feedback can act to enhance the hormone secretion ( + , positive feedback). (b) The endocrine organ

produces hormone 1, which acts on a second endocrine gland to release hormone 2. In turn, hormone 2 acts dually on the target tissue to induce a response and feeds back negatively onto the original endocrine organ to inhibit further release of hormone 1. This model is illustrative of the axes between the anterior pituitary and the peripheral end-organ targets.

Positive feedback

establish positive feedback that is only terminated by delivery of the baby. The role of oxytocin in the suckling–milk ejection reflex is similar – a positive feedback loop that is only broken by cessation of the baby’s suckling.

Under certain, more unusual, circumstances, hormone feedback enhances, rather than inhibits, the initial response. This is called positive feedback (an example is shown alongside the more usual negative feedback in Figure 1.4a). This is intrinsically unstable. However, in some biological systems it can be transiently beneficial: for instance, the action of oestrogen on the pituitary gland to induce the ovulatory surge of LH and FSH (see Chapter 7); or during childbirth, stretch receptors in the distended vagina and nerves to the brain stimulate oxytocin release. This hormone causes the uterus to contract, further activating the stretch receptors to

Inhibitory control The secretion of some hormones is under inhibitory as well as stimulatory control. Somatostatin, a hypothalamic hormone, prevents the secretion of GH, such that when somatostatin secretion is dim­ inished, GH secretion is enhanced. Prolactin is similarly controlled, under tonic inhibition from dopamine.

Chapter 1: Overview of endocrinology  /  13

Box 1.5  Endocrine cycles Circadian = 24-h cycle • Circa = about, dies = day Ultradian  24-h cycle • E.g. menstrual cycle

Endocrine rhythms Many of the body’s activities show periodic or cyclical changes (Box 1.5). Control of these rhythms commonly arises from the nervous system, e.g. the hypothalamus. Some appear independent of the environment, whereas others are coordinated and ‘entrained’ by external cues (e.g. the 24-h light/dark cycle that becomes temporarily disrupted in jetlag). Cortisol secretion is maximal between 0400h and 0800h as we awaken and minimal as we retire to bed. In contrast, GH and prolactin are secreted maximally ∼1 h after falling asleep. Clinically, this knowledge of endocrine rhythms is important as investigation must be referenced according to hour-by-hour and day-to-day variability. Otherwise, such laboratory tests may be invalid or, indeed, misleading.

Endocrine disorders The chapters in Part 2 largely focus on organspecific endocrinology and associated endocrine disorders. Diabetes in Part 3, incorporating obesity, has now become its own specialized branch of endocrinology. Nevertheless, it is possible to regard all endocrine abnormalities as disordered, too much or too little production of hormone. Some clinical features can occur because of compensatory overproduction of hormones. For example, Addison disease is a deficiency of cortisol from the adrenal cortex (see Chapter 6), which reduces negative feedback on ACTH production at the anterior pituitary. ACTH rises and stimulates melanocytes in the skin to increase pigmentation – a cardinal sign of Addison disease. Imbalanced hormone production may occur when a particular enzyme is missing because of a

genetic defect. For example, in congenital adrenal hyperplasia, the lack of 21-hydroxylase causes failure to synthesize cortisol (see Chapter 6). Other pathways remain intact, leading to excess production of sex steroids that can masculinize aspects of the female body. Endocrine disorders may also arise from abnormalities in hormone receptors or downstream signalling pathways. The commonest example is type 2 diabetes, which arises in part from resistance to insulin action in target tissues (see Chapter 13). For those endocrine glands under regulation by the hypothalamus and anterior pituitary, disorders can also be categorized according to site. Disease in the end organ is termed ‘primary’. When the end organ is affected downstream of a problem in the anterior pituitary (either underactivity or overactivity), it is secondary, while in tertiary disease, the pathology resides in the hypothalamus. Like in other specialties, tumourigenesis impacts on clinical endocrinology. Most commonly, these tumours are sporadic and benign, but they may oversecrete hormones, and are described in the appropriate organ-specific chapters in Part 2. However, endocrine tumourigenesis may also form part of recognized multiorgan clinical syndromes. These are described in Chapter 10.

Key points • Endocrinology is the study of hormones and forms one of the body’s major communication systems • A hormone is a chemical messenger, commonly distributed via the circulation, that elicits specific effects by binding to a receptor on or inside target cells • The three major types of hormones are peptides, and the derivatives of amino acids and cholesterol • Negative and, occasionally, positive feedback, and cyclical mechanisms operate to regulate hormone production, commonly as part of complex multiorgan systems or axes • Clinical endocrine disorders usually arise through too much, too little or disordered hormone production

14

CHAPTER 2

Cell biology and hormone synthesis Key topics ■ Chromosomes, mitosis and meiosis

15

■ Making a protein or peptide hormone

16

■ Making a hormone derived from amino

acids or cholesterol

20

■ Hormone transport

24

■ Key points

26

Learning objectives ■ To appreciate the organization, structure and function of DNA ■ To understand mitosis and meiosis ■ To understand protein synthesis and peptide hormone

production ■ To understand the function of enzymes and how enzyme

cascades generate steroid and amino acid-derived hormones This chapter aims to introduce some of the basic principles that are needed to understand later chapters

Essential Endocrinology and Diabetes, Sixth Edition. Richard IG Holt, Neil A Hanley. © 2012 Richard IG Holt and Neil A Hanley. Publlished 2012 by Blackwell Publishing Ltd.

Chapter 2: Cell biology and hormone synthesis  /  15

This chapter introduces five major themes: chromosomes and DNA; protein (and peptide) hormone synthesis; hormones derived from amino acids; steroid hormones and vitamin D; and hormone transport in the circulation. How hormones exert their actions is covered in Chapter 3. The human genome is made up of deoxyribonucleic acid (DNA), assembled into 46 chromosomes, and resides in the nucleus (Box 2.1). The DNA contains the ‘blueprints’, called genes, for synthesizing proteins. There are approximately 30,000 human genes. Each gene serves as the template for generating many copies of messenger ribonucleic acid (mRNA) by a process called gene expression that amplifies the information contained in a single gene into the building blocks for many replica proteins. Specific proteins define the phenotype of a particular cell type (e.g. a thyroid cell that synthesizes thyroid hormone); more commonplace proteins carry out basic functions, e.g. the metabolic processes common to all cells. Proteins on the

Box 2.1  The structure of DNA • A molecule of deoxyribose (a 5-carbon sugar) is linked covalently to one of two types of nitrogenous bases: ° Purine – adenine (A) or guanine (G) ° Pyrimidine – thymine (T) or cytosine (C) ° The base plus the sugar is termed a ‘nucleoside’, e.g. adenosine • The addition of a phosphate group to a nucleoside creates a nucleotide, e.g. adenosine mono-, di- or tri-phosphate (according to how many phosphate groups have been added) • Phosphodiester bonds polymerize the nucleotides into a single strand of DNA • Two strands, running in opposite directions, 5 prime (5′; upstream) to 3′ (downstream) assemble as a double helix: ° Hydrogen bonds form between the strands, between the base pairs A–T and G–C • ∼3 billion base pairs comprise the human genome

cell surface act as receptors that initiate intracellular signalling, which in turn is reliant on proteins that function as enzymes. Eventually, signalling information reaches the nucleus and the proteins within it, called transcription factors. These latter proteins bind or release themselves from areas of DNA around genes to determine whether a gene is switched on (‘expressed’, when mRNA is transcribed) or silenced.

Chromosomes, mitosis and meiosis Genomic DNA in most human cells is packaged into chromosomes by being wrapped around proteins called histones – the DNA–histone complex is referred to as chromatin. There are 22 pairs of ‘autosomes’ and two sex chromosomes – two Xs in females, one X and a Y in males. This composition makes females 46,XX and males 46,XY. Distinct chromosomes are only apparent when they are lined up in preparation for cell division, either ‘mitosis’ or ‘meiosis’ (Figure 2.1). Mitosis generates two daughter cells, each with a full complement of 46 chromosomes, and occurs ∼1017 times during human life. Meiosis creates the gametes (i.e. spermatozoan or ovum), each with 23 chromosomes so that full diploid status is reconstituted at fertilization. Several chromosomal abnormalities can result in endocrinopathy. During meiosis, if a chromosome fails to separate properly from its partner or if migration is delayed, a gamete might result that lacks a chromosome or has too many. Thus, it is easy to appreciate Turner syndrome (45,XO), where one sex chromosome is missing; or Klinefelter syndrome (47,XXY), where there is an extra X. Similarly, breaks and rejoining across or within chromosomes produce unusual ‘derivative’ chromosomes or ones with duplicated or deleted regions (see Figure 4.4). If such events occur close to genes, function can be disrupted, e.g. congenital loss of a hormone. Duplication can be equally significant; on the X chromosome, a double dose of a region that includes the dosage-sensitive sex reversal, adrenal hypoplasia critical region gene 1 (DAX1) causes female development in a 46,XY fetus.

16  /  Chapter 2: Cell biology and hormone synthesis

(a)

Mitosis:

retains the full complement of 46 chromosomes to generate two diploid daughter cells

Interphase Nuclear membrane Nucleolus visible No chromosomes DNA synthesis (‘S’ phase)

Prophase Spindle formation Chromosomes condense

Prometaphase Nuclear membrane dissolves Chromosomes migrate centrally

Metaphase Chromosomes centrally positioned at ‘metaphase plate’

‘M’ phase

Anaphase Chromatids separate as centromere splits

Cytokinesis Cell divides into two daughter cells

Telophase Chromosomes separate to each pole and start to decondense Nuclear membrane reforms Cytoplasm starts to divide

Figure 2.1  Cell division. Prior to mitosis and meiosis the cell undergoes a period of DNA synthesis (‘S’ phase) so that the normal diploid status of DNA (2n) temporarily becomes 4n. (a) The stages of mitosis result in each daughter cell containing diploid 2n quantities of DNA. (b, opposite) Meiosis is split into two stages, each of which comprises prophase, prometaphase, metaphase, anaphase and telophase. During prophase of meiosis I, the maternally and paternally derived chromosomes align to allow crossing over (‘recombination’), a

critical aspect of genetic diversity. The two sister chromatids do not separate, so that the secondary oocyte and spermatocytes each contains 2n quantities of DNA. During the second stage of meiosis, separation of the chromatids results in haploid cells (n). In males, meiosis is an equal process resulting in four spermatids. In contrast, in females, only one ovum is produced from a primary oocyte, smaller polar bodies being extruded at both stages of meiosis.

Making a protein or peptide hormone

base pairs. Transcription factors bind to these elements either to promote (i.e. turn on) or to repress (i.e. turn off) the production of mRNA by RNA polymerase, the enzyme that ‘reads’ the DNA code. Commonly, the signal that recruits RNA polymerase to the DNA occurs at a ‘TATA’ box, a short run of adenosines and thymidines, ∼30 base pairs upstream of exon 1 (Figure 2.2) or an area rich in G and C residues. Superimposed on this, gene expression can be further increased or diminished by more cell- or tissue-specific transcription factors potentially

Gene transcription and its regulation The production of mRNA from a gene is called transcription. Within most genes, the stretches of DNA that encode protein, called exons, are separated by variable lengths of non-coding DNA called introns (Figure 2.2). Upstream of the first exon is the 5′ flanking region of the gene, which contains multiple promoter elements, usually within a few hundred

Chapter 2: Cell biology and hormone synthesis  /  17

(b) Meiosis:

halves the chromosomal complement to generate haploid daughter cells each with 23 chromosomes

Female

Primary oocyte First polar body

Secondary oocyte

Second polar body

Mature ovum

Mitotic division of the diploid (2n) germ cells; oogonia (ovary) and spermatogonia (testis)

DNA synthesis (4n) in differentiated cells

Male

Primary spermatocyte

Meiosis I

2n DNA cells

Secondary spermatocytes

Meiosis II

Haploid (n) cells

Spermatids

Figure 2.1  (Continued)

binding to more distant stretches of DNA (either ‘enhancer’ or ‘repressor’ elements). For instance, the transcription factor, steroidogenic factor-1 (SF-1), turns on many genes specific to the adrenal cortex and gonad; when SF-1 is absent, both organs fail to form. These alterations to expression are dependent on the exact DNA sequence recognized by specific proteins. There is another layer of complexity governing how genes are expressed. Epigenetics is the study of how gene expression is regulated by mechanisms beyond the precise DNA sequence, e.g. by methylation of the DNA around genes, which tends to silence expression; or by modifications to histones, such as acetylation or methylation, which alter the chromatin structure and make stretches of DNA containing genes accessible or inaccessible to transcription factors. Acetylation tends to open up the structure, facilitating gene expression, whereas methylation tends to close it down, silencing transcription. Genomic imprinting is an epigenetic phenomenon involving DNA methylation and

modifications to histones such that gene expression varies according to which parent the particular chromosome came from. RNA contains ribose sugar moieties rather than deoxyribose. RNA polymerase ‘sticks’ ribonucleotides together to generate a single strand of mRNA that correlates to the DNA code of the gene, except that in place of thymidine, a very similar nucleoside, uridine, is incorporated. The initial mRNA strand (pre-mRNA) is processed so that intronic gene regions are excluded and only the exonic sequences are ‘spliced’ together. Not all exonic regions encode protein; stretches at either end constitute the 5′ and 3′ untranslated regions (UTRs) (Figure 2.2). Within the 3′ UTR, mRNA transcription is terminated by a specific purine-rich motif, the polyadenylation signal, ∼20 base pairs upstream of where the mRNA gains a stretch of adenosine residues. This poly-A tail provides stability as the mRNA is moved from the nucleus to the cytoplasm for translation into protein.

18  /  Chapter 2: Cell biology and hormone synthesis

Intron 1

5’ flanking region

Intron 2

3’ flanking region

TATA Exon 2

Exon 1

Gene (DNA) Enhancer element

Pre-mRNA

Exon 3

Basal promoter

Stop PolyA codon signal

Exon 1

Exon 2

Exon 3 AAAAAA

Intron 1

Exon 1

Exon 2

Start codon (AUG) Peptide

Exon 1

Intron 2 3’ UTR

5’ UTR Spliced mRNA

Repressor element

Exon 2

Exon 3 AAAAAA Stop codon Ex3

Met

Figure 2.2  Schematic representation of a gene, transcription and translation. In this example, the gene comprises three exons with an enhancer element in the 5′ flanking region and a repressor element in the 3′ flanking region. UTR, untranslated region. Met, methionine (encoded by the start codon).

Translation into protein mRNA is transported to the ribosomes, where protein synthesis occurs by translation (Figure 2.3a). The ribosomes are attached to the outside of the endoplasmic reticulum (ER), leading to the description of rough ER. The ribosome is an RNA–protein complex that ‘reads’ the mRNA sequence. On the first occasion that sequential A–U–G nucleotides are encountered (corresponding to ATG in the genomic DNA), translation starts (see Figure 2.2). From this point, every three nucleotides represent an amino acid. This nucleotide triplet is called a codon, AUG being a start codon that specifies the amino acid methionine. Similarly, translation continues until a ‘stop’ codon is encountered (UAA, UGA or UAG). By understanding these normal events of gene transcription and protein translation, it becomes

possible to appreciate how mutations (sequence errors) in the genomic DNA lead to a miscoded, and consequently malfunctional, protein (Box 2.2). An entire gene may be missing (‘deleted’) or duplicated. An erroneous base pair in the promoter region may impair a critical transcription factor from binding. A similar error in an exonic coding sequence might translate a different amino acid or even create a premature stop codon. Small deletions or insertions of one or two base pairs throw the whole triplet code out of frame. A mutation at the boundary between an intron and an exon can prevent splicing so that the intron becomes included in the mature mRNA. All of these events affect endocrinology either as congenital defects (i.e. during early development so that the fault is present in the genome of many cells) or as acquired abnormalities later in life, potentially predisposing to the formation of an endocrine tumour (see Chapter 10).

Chapter 2: Cell biology and hormone synthesis  /  19

(a)

(b)

Peptide hormone synthesis

Steroid hormone synthesis

Nucleus

Mitochondrion Rough endoplasmic reticulum

Mitochondrion

mRNA

Cholesterol

Lysosome Lipid droplet

Golgi complex Smooth endoplasmic reticulum

Secretory granules

Secretion

Microtubules Exocytosis of granule Microfilaments

Lysosome Golgi complex Capillary

Figure 2.3  (a) Peptide hormone-synthesizing and (b) steroid hormone-synthesizing cells. In (b) cholesterol enters the cell via the low-density lipoprotein receptor which is internalized.

Post-translational modification of peptides Some polypeptides can function as hormones after little more than removal of the starting methionine, e.g. thyrotrophin-releasing hormone (TRH), which consists of only three amino acids. Others undergo further modification or potentially fold together as different subunits of a hormone, e.g. luteinizing hormone (LH). Peptides with any degree of complexity fold into three-dimensional structures, which may contain helical or pleated domains. These shapes provide stability and affect how one protein interacts with another (e.g. how a hormone might bind to its receptor). For hormones that require secretion out of the cell, additional modifications are common and

important (Figure 2.4). The precursor peptide, called a pre-prohormone, carries a lipophilic signal peptide at the amino terminus. This sequence is recognized by channel proteins so that the immature peptide can cross the ER membrane. Once inside the ER, the signal peptide is excised in preparation for other post-translational changes (Figure 2.4a–d). Disulphide bridges are formed in certain proteins (e.g. growth hormone or insulin; Figure 2.4a and c). Certain carbohydrates may be added to form glycoproteins (Figure 2.4d). Some prohormones (e.g. pro-opiomelanocortin and proglucagon) need processing to give rise to several final products, whereas others are assembled as a combination of distinct peptide chains, each synthesized from different genes, e.g. thyroid-stimulating hormone (TSH).

20  /  Chapter 2: Cell biology and hormone synthesis

Box 2.2  Genetic, genomic and epigenetic abnormalities that can result in endocrinopathy Abnormalities in DNA (genetic) • Base substitution – swapping different nucleotides • Insertion or deletion – alters frame if exonic and not a multiple of three Chromosomal abnormalities (genomic) • Numerical – three copies as in Down syndrome (trisomy 21) • Structural: ° Inversions – region of a chromosome is turned upside down ° Translocations – regions swapped between chromosomes ° Duplications – region of a chromosome is present twice ° Deletions – region of a chromosome is excised and lost Imprinting abnormalities (epigenetic) • Methylation – altered methylation changing local gene expression, such as Beckwith– Wiedemann syndrome with neonatal hypoglycaemia or transient neonatal diabetes associated with overexpression of the gene called ZAC • Structural chromosomal abnormalities (as above) can also cause imprinting errors

The completed protein is then packaged into membrane-bound vesicles, which may contain specific ‘endopeptidases’. These enzymes are responsible for final hormone activation – cleaving the ‘pro-’portion of the protein chain, as occurs with the release of C-peptide and insulin (Figure 2.4c). Such post-translational modifications are essential stages in hormone synthesis (Box 2.3). Storage and secretion of peptide hormones Endocrine cells usually store newly synthesized peptide hormone in small vesicles or secretory gran-

Box 2.3  Role of post-translational modifications Post-translational modifications are important so that: • Great diversity of hormone action is generated from a more limited range of encoding genes • Active hormone is saved for its intended site of action • The synthesizing cell is protected from a barrage of its own hormone action

ules. Movement of these vesicles to a position near the cell membrane is influenced by two types of filamentous structure: microtubules and microfilaments (see Figure 2.3a). Secretion of the stored hormone tends to be rapid but only occurs after appropriate stimulation of the cell. Whether this is hormonal, neuronal or nutritional, it usually involves a change in cell permeability to calcium ions. These divalent metal ions are required for interaction between the vesicle and plasma membrane, and for the activation of enzymes, microfilaments and microtubules. The secretory process is called exocytosis (see Figure 2.3a). The membrane of the storage granule fuses with the cell membrane at the same time as vesicular endopeptidases are activated so that active hormone is expelled into the extracellular space from where it enters the blood vessels. The vesicle membrane is then recycled within the cell.

Making a hormone derived from amino acids or cholesterol In addition to peptides or proteins, hormones can also be synthesized by sequential enzymatic modification of the amino acids tyrosine and tryptophan, or of cholesterol. Enzyme action and cascades Enzymes can be divided into classes according to the reactions that they catalyze (Table 2.1). In endocrinology, they frequently operate in cascades where the

Chapter 2: Cell biology and hormone synthesis  /  21

Signal sequence

5'

Hormone-encoding sequence AAAA

mRNA

3'

Translation on ribosomes attached to the outer membrane of the endoplasmic reticulum, cleavage of the signal ('pre-') sequence, potential glycosylation ±CHO

±CHO

Endoplasmic reticulum

+

Signal peptide

Hormone

CHO B

Mature hormone or Pro-hormone

Polyprotein

C

Pro-hormone Orientation of functional peptides

Multiple processing

Disulphide bond formation and/or cleavage of aminoterminal pro-hormone sequence

CHO Alpha

A

CHO

Separate genes for alpha and beta subunits

A

Subunit assembly (± further glycosylation)

S S

S S

CHO

Beta

B

Golgi Secretory granules

Cleavage S

S

or Growth hormone or parathyroid hormone (a)

CHO

A

Pro-opiomelanocortin, glucagon, somatostatin and calcitonin precursors (b)

S

S

S

S B

CHO

+ C-peptide CHO

Insulin

(c)

Secreted hormone

Alpha Beta

CHO

Thyrotrophin, gonadotrophins, human chorionic gonadotrophin

Examples

(d)

Figure 2.4  Post-translational modifications of peptide hormones. Four types are shown. (a) Simple changes such as removal of the amino terminal ‘pro-’extension prior to secretion (e.g. parathyroid hormone) or the addition of intra-chain disulphide bonds (e.g. growth hormone). (b) Multiple processing of a ‘polyprotein’ into a number of different peptide hormones (e.g. proopiomelanocortin can give rise to adrenocorticotrophic hormone plus melanocytestimulating hormone and β-endorphin). (c) Synthesis

of insulin requires folding of the peptide and the formation of disulphide bonds. The active molecule is created by hydrolytic removal of a connecting (C)-peptide, i.e. proinsulin gives rise to insulin plus C-peptide in equimolar proportion. (d) Synthesis of larger protein hormones (e.g. thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone and human chorionic gonadotrophin) from two separate peptides that complex together. The four hormones share the same α-subunit with a hormonespecific β-subunit.

product of one reaction serves as the substrate for the next. Most simplistically, enzyme action is achieved by protein–protein interaction between the sub­ strate and the enzyme at the latter’s ‘active site’. This causes a modification to the substrate to form a product which no longer binds to the active site and is thus released. Other macromolecules bind else-

where to the enzyme and function as co-factors, adding more complex regulation to the biochemical reaction. Patients can present with many endocrine syndromes because of loss of enzyme function. For instance, gene mutation might lead to substitution of an amino acid at a key position of an enzyme’s

22  /  Chapter 2: Cell biology and hormone synthesis

Table 2.1  Definition and classification of enzymes Definition An enzyme is a biological macromolecule – most frequently a protein – that catalyzes a biochemical reaction Catalysis increases the rate of reaction, e.g. the disappearance of substrate and generation of product Enzyme action is critical for the synthesis of hormones derived from amino acids and cholesterol Classification Enzyme

Catalytic function

Example (and relevance)

Hydrolases

Cleavage of a bond by the addition of water

Cytochrome P450 11A1/cholesterol side-chain cleavage (CYP11A1; early step in steroid hormone biosynthesis)

Lyases

Removal of a group to form a double bond or addition of a group to a double bond

Cytochrome P450 17α-hydroxylase/17–20 lyase (CYP17A1; step in synthesis of steroid hormones other than aldosterone)

Isomerases

Intramolecular rearrangments

3β-hydroxysteroid dehydrogenase/delta 4,5 isomerase isoforms (HSD3B; step in synthesis of all major steroid hormones)

Oxidoreductase

Oxidation and reduction

11β-hydroxysteroid dehydrogenase isoforms (HSD11B; inter-conversion of cortisol and cortisone)

Ligases or synthases

Joins two molecules together

Thyroid peroxidase (TPO; step in synthesis of thyroid hormone)

Transferases

Transfer of a molecular group from substrate to product

Phenol ethanolamine N-methyl transferase (PNMT; conversion of norepinephrine to epinephrine)

active site. The three-dimensional structure might be affected so significantly that the substrate may no longer convert to product. In the enzyme cascade that synthesizes cortisol, this causes various forms of congenital adrenal hyperplasia (CAH) (see Chapter 6). Understanding the biochemical cascade allows accurate diagnosis as the substrate builds up in the circulation and its excess can be measured, e.g. by immunoassay or mass spectrometry (see Chapter 4).

Synthesizing hormones derived from amino acids The amino acid tyrosine can be modified by sequential enzyme action to give rise to several hormones (Box 2.4). The precise synthetic pathways for dopamine and catecholamines are described in

Box 2.4  Hormones derived from tyrosine • Thyroid hormones: sequential addition of iodine and coupling of two tyrosines together (see Chapter 8) • Adrenomedullary hormones: hydroxylation steps and decarboxylation to form dopamine and catecholamines (see Chapter 6) • Hypothalamic dopamine formed by hydroxylation and decarboxylation (see Chapter 5)

Chapter 6 (Figure 6.11) and for thyroid hormones in Chapter 8 (Figures 8.3 and 8.4). Melatonin, important in endocrine circadian rhythms with a new link to type 2 diabetes (see Chapter 5), is gener-

Chapter 2: Cell biology and hormone synthesis  /  23

ated from the amino acid tryptophan via synthesis of the neurotransmitter serotonin. Synthesizing hormones derived from cholesterol Steroid hormones are generated by enzyme cascades that modify the four-carbon ring structure of cholesterol (see Figure 2.6). The precise sequence of enzymes determines which steroids are generated as the final product (Box 2.4). In addition to making steroid hormones, cholesterol is a critical building block of all mammalian cell membranes and the starting point for synthesizing vitamin D, which functions, and can be classified, as a hormone (see Chapter 9). Cholesterol is acquired in approximately equal measure from the diet and de novo synthesis (mostly in the liver; Box 2.5). From the diet, cholesterol is delivered to cells as a complex with low-density lipoprotein (LDL–cholesterol). Intracellular uptake is via the cell-surface LDL receptor and endocytosis (see Figure 2.3b). De novo biosynthesis commences with co-enzyme A (CoA), itself synthesized from pantothenate, cysteine and adenosine, and proceeds via hydroxymethylglutaryl co-enzyme A (HMGCoA) and mevalonic acid (Figure 2.5). The rate-limiting step is the reduction of HMGCoA by the enzyme HMGCoA reductase. Pharmacological inhibition of this enzyme to treat hypercholesterolaemia and lessen cardiovascular disease has led to the most widely prescribed drug family in the world (the ‘statins’) and the award of the Nobel Prize to Michael Brown and Joseph Goldstein (see Table 1.2).

Box 2.5  Hormones derived from cholesterol Cholesterol derived from the diet and de novo synthesis used to synthesize: • Vitamin D (see Chapter 10) • Steroid hormones: ° Adrenal cortex: aldosterone, cortisol and sex steroid precursors (see Chapter 6) ° Testis: testosterone (see Chapter 7) ° Ovary and placenta: oestrogens and progesterone (see Chapter 7)

In steroidogenic cells, cholesterol is largely deposited as esters in large lipid-filled vesicles (see Figure 2.3b). Upon stimulation, cholesterol is released from its stores and transported into the mitochondria, a process that is facilitated by the steroid acute regulatory (StAR) protein in the adrenal and gonad and by the related protein, start domain containing 3 (STARD3), in the placenta. The first step in the synthesis of a steroid hormone is the rate-limiting conversion of cholesterol to pregnenolone. Pregnenolone then undergoes a range of further enzymatic modifications in the mitochondria or the ER to make active steroid hormones. Nomenclature of steroidogenic pathways Figure 2.6 shows a generic representation of human steroid hormone biosynthesis. Many of the enzymes that catalyze steps in these pathways are encoded by the cytochrome P450 (CYP) family of related genes that is also critical for hepatic detoxification of drugs. Some of the enzymes are important in both the adrenal cortex and gonad (e.g. CYP11A1), whereas others are restricted and thus create the distinct steroid profiles of each tissue (e.g. CYP21A2, needed for cortisol and aldosterone biosynthesis, is very largely limited to the adrenal cortex). Historically, the enzymes have been named according to function (e.g. hydroxylation) at a specific carbon atom, with a Greek letter indicating orientation above or below the four-carbon ring structure (e.g. 17α-hydroxylase attaches a hydroxyl group in the alpha position to carbon 17; Figure 2.6). The common names used for steroids also adhere to a loose convention. The suffix -ol indicates an important hydroxyl group, as in cholesterol or cortisol, whereas the suffix -one indicates an important ketone group (testosterone). The extra presence of -di, as in -diol (oestradiol) or -dione (androstenedione), reflects two of these groups, respectively; ‘-ene’ (androstenedione) within the name indicates a significant double bond in the steroid nucleus. Storage of steroid hormones Unlike cells making peptide hormones, most steroid-secreting cells do not store hormones but

24  /  Chapter 2: Cell biology and hormone synthesis

Acetyl CoA

Acetoacetyl CoA

HMG-CoA synthase 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) HMG-CoA reductase Mevalonic acid Mevalonate kinase Mevalonate-5-phosphate Phosphomevalonate kinase Mevalonate-5-pyrophosphate Isopentenyl-5-pyrophosphate (PP) Geranyl PP

Mevalonate-5-pyrophosphate decarboxylase

Farnesyl PP synthase

Farnesyl PP Squalene synthase Squalene A series of 21 reactions CHOLESTEROL Figure 2.5  Synthesis of cholesterol. Step is catalyzed by the enzyme thiolase; otherwise the enzymes are shown to the right of the cascade. The individual steps between squalene and cholesterol have been omitted.

synthesize them as required. As a consequence, there is a slower onset of action for steroid hormones following the initial stimulation of the steroidogenic organ.

Hormone transport Most peptide hormones are hydrophilic, so they generally circulate free in the bloodstream with little or no association with serum proteins. In contrast, steroid hormones and thyroid hormones circulate bound to proteins because, like cholesterol, they are hydrophobic. There are relatively specific transport proteins for many of the steroid hormones, e.g. cortisol-binding globulin (CBG) and sex hormone-binding globulin (SHBG), as well

as for thyroid hormones [thyroxine-binding globulin (TBG)]. Many hormones also loosely associate with other circulating proteins, especially albumin. The equilibrium between protein-bound and unbound (‘free’) hormone determines activity, as only free hormone readily diffuses into tissues. This is relevant to many hormone assays where total hormone is measured. Results may be markedly altered by changes in the concentration of binding protein, but the change in free hormone concentration may be very small and biological activity remains unaltered. For instance, women on the combined oral contraceptive pill have raised serum CBG and increased total cortisol; however, free cortisol is unaltered.

Chapter 2: Cell biology and hormone synthesis  /  25

(a)

22

21

23

20 12 11 1

10

A HO

3

4

C

19

2

5

9

B

8

18 13

26

24 17

25

16

D

27

15

14

7 6

Cholesterol CYP11A1 Cholesterol side chain cleavage

CH3

CH3

O

O OH

O CYP17A1 17α-hydroxylase

CYP17A1 17–20 lyase

HO 17-OH Pregnenolone

HO Pregnenolone CH3

HSD3B 3β-hydroxysteroid dehydrogenase

DHEA CH3

HSD3B

O

HO HSD3B

O OH

O

CYP17A1 17α-hydroxylase O

O 17-OH Progesterone

Progesterone CH2OH O

(b)

CYP21A2 21-hydroxylase

O Deoxycorticosterone CYP11B2 Aldosterone synthase HO

CH2OH O

CH2OH O OH

CYP21A2 21-hydroxylase

O

CYP11B2 Aldosterone synthase

Androstenedione

HSD17B

CYP11B1 11β-hydroxylase

Cortisol

CH2OH O OH

OH

17β-hydroxysteroid dehydrogenase

O

O Corticosterone

(c)

11 Deoxycortisol

HO

O

O

Testosterone

CYP19A1 Aromatase

HO

OH

Oestradiol

CH2OH CHO

O

HO

O Aldosterone

Figure 2.6  Overview of the major steroidogenic pathways. Yellow shading indicates the enzymatic change since the last step. The enzymes are shown by proper name and common name according to their action. Note some enzymes perform multiple reactions (e.g. CYP17A1 acts as both a hydroxylase and lyase) and some reactions are performed by multiple enzyme isoforms (e.g. HSD3B exists as HSD3B1 and HSD3B2). For 17-OH progesterone, the

enzymatic change illustrated is that catalyzed by HSD3B activity rather than CYP17A1. The simplified pathways are grouped into three blocks: (a) common to both the adrenal cortex and the gonad; (b) adrenocortical steroidogenesis; and (c) pathways characteristic of the testis (testosterone) or ovary and placenta (oestradiol). OH, hydroxy; DHEA, dehydroepiandrosterone.

26  /  Chapter 2: Cell biology and hormone synthesis

Key points • Mutations in DNA and chromosomal abnormalities cause congenital and sporadic endocrine disease • Meiosis is central to reproductive endocrinology • Genes are stretches of DNA responsible for encoding protein • Many protein hormones are synthesized as prohormones requiring post-translational modification and processing before they become active

• Enzyme cascades synthesize hormones derived from amino acids and cholesterol • Unlike peptide hormones, steroid hormones are not stored in cells but made ‘on demand’ • Many peptide hormones are free in the circulation, unlike steroid or thyroid hormones, which associate with binding proteins

27  

CHAPTER 3

Molecular basis of hormone action Key topics ■ Cell-surface receptors

28

■ Tyrosine kinase receptors

31

■ G-protein–coupled receptors

34

■ Nuclear receptors

40

■ Key points

47

Learning objectives ■ To understand the principles of hormone–receptor interaction ■ To understand the biology of the different families of

hormone receptors ☐  Tyrosine kinase receptors and associated signalling

pathways ☐ G-protein–coupled receptors and associated signalling

pathways ☐ Nuclear receptors and their influence on gene expression ■ To appreciate the role of transcription factors that are

important in endocrine development and function ■ To appreciate how abnormalities in hormone receptors or

their downstream signalling can cause endocrinopathy This chapter describes the key events that occur within the cell following stimulation by hormone

Essential Endocrinology and Diabetes, Sixth Edition. Richard IG Holt, Neil A Hanley. © 2012 Richard IG Holt and Neil A Hanley. Publlished 2012 by Blackwell Publishing Ltd.

28  /  Chapter 3: Molecular basis of hormone action

Stage 1: Binding of hormone to receptor

Hormones act by binding to receptors. There are two superfamilies of hormone receptor: the cellsurface receptors and nuclear receptors, named according to their site of action, and which display characteristic features (Figure 3.1 and Box 3.1).

Historically, hormone–receptor interactions (Box 3.2) have been characterized using radiolabelled hormones and isolated preparations of receptors to define two properties:

Cell-surface receptors

• The hormone–receptor interaction is saturable (Figure 3.3) • The hormone–receptor interaction is reversible (Figure 3.4).

Cell-surface receptors comprise three components, each with characteristic structural features that reflect their location and function (Figure 3.2).

Hormone receptors

Superfamilies

Groups

Nuclear receptors

Cell-surface receptors

Linked to TK

Linked to G proteins

Subgroups

Adenylate cyclase

PLC

e.g. TRH e.g. TRH GnRH GnRH TSH Somatostatin LH TSH e.g. Insulin FSH LH IGF-I Cytokine receptors ACTH FSH which recruit TK Vasopressin Oxytocin Catecholamines Vasopressin e.g. Growth hormone Glucagon Angiotensin II Prolactin PTH Ca2+ Leptin Calcitonin PTHrP PTH PGE2 GHRH PTHrP

Growth factor receptors with intrinsic TK

Orphan and variant

Metabolites e.g. peroxisome proliferator-activated receptors (PPARs)

Figure 3.1  The different classes of hormone receptor. Some cell-surface receptors, e.g. the parathyroid hormone (PTH) receptor, can link to different G-proteins, which couple to either adenylate cyclase or phospholipase C (PLC). TK, tyrosine kinase; TRH, thyrotrophin-releasing hormone; GnRH, gonadotrophin-releasing hormone; TSH, thyroid-

Steroid/thyroid hormone

Vitamin e.g. calcitriol

stimulating hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; ACTH, adrenocorticotrophic hormone; PTHrP, parathyroid hormone-related peptide; PGE2, prostaglandin E2; GHRH, growth hormone releasing hormone; IGF-I, insulin-like growth factor I.

Chapter 3: Molecular basis of hormone action  /  29

Box 3.1  Some basic facts about hormone receptors • Tissue distribution of receptor determines the scope of hormone action: ° Thyroid-stimulating hormone (TSH) receptor largely limited to thyroid, therefore TSH action restricted to the thyroid ° Thyroid hormone receptor is widespread, therefore thyroid hormone action is diverse • Binding of hormone induces a conformational change in the receptor that initiates downstream signalling • Downstream signalling differs across cell types to produce potentially diverse hormonal effects • Control is exerted through the constant synthesis, degradation and localization of hormone receptors – most target cells have 2000–100,000 receptors for a particular hormone

Hormone receptor superfamilies • Water-soluble hormones (e.g. peptide hormones): ° Plasma membrane is impenetrable ° Cell-surface receptors transduce signal through membrane ° Activate intracellular signalling pathways ° Fast responses (seconds) as well as slow ones • Lipid-soluble hormones (e.g. steroid and thyroid hormones): ° Pass through plasma membrane ° Receptors function as transcription factors in the nucleus ° Activate or repress gene expression ° Tend to be relatively slow responses (hours–days)

NH2

Extracellular domain (hormone binding)

Hydrophobic transmembrane domain

Plasma membrane

Cytoplasmic domain (initiates intracellular signalling) COOH

Figure 3.2  A membrane-spanning cell-surface receptor. The hormone acts as ligand. The ligandbinding pocket in the extracellular domain is comparatively rich in cysteine residues that form internal disulphide bonds and repeated loops to ensure correct folding. For some hormones, e.g. growth hormone, this extracellular domain can circulate as a potential binding protein. Circulating

fragments of the thyroid-stimulating hormone receptor may be immunogenic for antibody formation in autoimmune thyroid disease. The α-helical membranespanning domain is rich in hydrophobic and uncharged amino acids. The C-terminal cytoplasmic domain either contains, or links to, separate catalytic systems, which initiate the intracellular signals after hormone binding.

30  /  Chapter 3: Molecular basis of hormone action

Half-maximal saturation

KD

Increasing concentration of labelled hormone Figure 3.3  Hormone–receptor systems are saturable. Increasing amounts of labelled hormone are incubated with a constant amount of receptor. The amount of bound labelled hormone increases as more is added until the system is saturated. At this point, further addition fails to increase the amount bound to receptors. The concentration of hormone that is required for half-maximal saturation of the receptors is equal to the dissociation constant (KD) of the hormone–receptor interaction.

Box 3.2  Binding characteristics of hormone receptors • High affinity: hormones circulate at low concentrations – receptors are like ‘capture systems’ • Reversible binding: one reason for the transient nature of endocrine responses • Specificity: receptors distinguish between closely related molecular structures

Using methodology similar to that for immunoassays (see Chapter 4), constant amounts of labelled hormone and receptor preparations can be incubated with increasing, known amounts of unlabelled hormone for a specified time. Separating and measuring the receptor-bound labelled fraction allows curves to be plotted and mathematical modelling of the hormone (H)–receptor (R) interac­ tion; e.g. whether it is conforms to the equation H + R  HR. Ultimately, these types of experi-

Amount of labelled hormone bound

Amount of labelled hormone bound

Maximum saturation

Add excess unlabelled hormone Time of incubation

Figure 3.4  Hormone–receptor interactions are reversible. Constant amounts of labelled hormone and receptors are incubated together for different times. The bound label increases with time until it reaches a plateau, when the bound and free hormone have reached a dynamic equilibrium. In a dynamic equilibrium, hormone continually associates and dissociates from its receptor. By adding excess unlabelled hormone, competition with the labelled hormone is established for access to the receptors. Consequently, the amount of bound labelled hormone decreases with extended incubation (dashed line).

ment can allow estimation of the number of hormone receptors present on each target cell. Stage 2: Signal transduction When a hormone binds to a cell-surface receptor, the cascade of cytoplasmic responses is mediated through protein phosphorylation by kinase enzymes or the generation of ‘second messengers’ via coupling to guanine (‘G’) proteins. Signalling through either process amplifies the hormone response as many protein phosphorylation events or second messenger molecules are produced for each hormone–receptor interaction. They also distinguish the two major groups of cell-surface receptor: tyrosine kinase receptors and G-protein–coupled receptors (Figure 3.1 and Box 3.3). Protein phosphorylation is a key molecular switch. Approximately 10% of proteins are phosphorylated at any given time in a mammalian cell. The phosphate group is donated from ATP during catalysis by the ‘kinase’ enzyme. It is accepted by the polar hydroxyl group of serine, threonine or

Chapter 3: Molecular basis of hormone action  /  31

Box 3.3  Categories of cell-surface receptors Tyrosine kinase receptors • Signal via phosphorylation of the amino acid, tyrosine G-protein–coupled receptors • Activate or inhibit adenylate cyclase and/or phospholipase C (PLC) • Signal via second messengers: cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3), diacylglycerol (DAG) and intracellular calcium • Signal via phosphorylation of serine and threonine amino acids

Receptors with intrinsic tyrosine kinase activity Intrinsic TK receptors autophosphorylate upon binding of the appropriate hormone. This group includes the receptor for insulin and those for epidermal growth factor (EGF), fibroblast growth factor (FGF) and insulin-like growth factor I (IGF-I). The EGF and FGF receptors exist as monomers that dimerize upon hormone binding to activate tyrosine phosphorylation. Those for insulin and IGF-I exist in their unoccupied state as preformed dimers. The signalling pathways for all these receptors are heavily involved in cell growth and proliferation. Insulin signalling pathways

tyrosine (Figure 3.5a) and causes a conformational change in the three-dimensional shape of the protein (Figure 3.5b). In many signalling pathways, the phosphorylated protein can also act as a kinase and phosphorylate the next protein in the sequence. In this way, a phosphorylation cascade is generated, which relays and amplifies the intracellular signal generated by the hormone binding to its receptor (Figure 3.5c).

Tyrosine kinase receptors Phosphorylation of tyrosine kinase (TK) receptors can occur through: • Intrinsic TK activity located in the cytosolic domain of the receptor; or • Separate TKs recruited after receptor activation (see Figure 3.1). By either mechanism, conformational change induced by phosphorylation creates ‘docking’ sites for other proteins. Frequently, this occurs via conserved motifs within the target protein, known as ‘SH2’ or ‘SH3’ domains. These domains may be involved in the activation of downstream kinases or they may stabilize other signalling proteins within a phosphorylation cascade.

The dimerized insulin receptor (IR) is composed of two α- and two β-subunits linked by a series of disulphide bridges (Figure 3.6; see Chapter 11). The earliest response to insulin binding is autophosphorylation of the cytosolic domains of the βsubunit. The activated receptor then phosphorylates two key intermediaries, insulin receptor substrate (IRS) 1 or 2, which are thought to be essential for almost all biological actions of insulin. IRS1 has many potential tyrosine phosphorylation sites, at least eight of which are phosphorylated by the activated IR. Multiple phosphorylation of IRS1 or 2 leads to the docking of several proteins with SH2 domains, and the activation of divergent intracellular signalling. For example, docking of phosphatidylinositol3-kinase (PI3-kinase) leads to deployment of the glucose transporter (GLUT) family members. For instance, in adipose tissue and muscle, GLUT-4 translocates from intracellular vesicles to the cell membrane, allowing glucose uptake into the cell. The mitogenic effects of insulin are mediated via a different intracellular pathway. Activated IRS1 docks with the SH2/SH3 domains of the type 2 growth factor receptor-bound (Grb2) protein. This adaptor protein links IRS1 to the son of sevenless (SoS) protein and, ultimately, to activation of the mitogen-activated protein kinase (MAPK) pathway, leading to gene expression that promotes mitosis and growth.

32  /  Chapter 3: Molecular basis of hormone action

(a)

Amino acids that can be phosphorylated. H

H

H

C

CO2H

CH2

CH

CH3

OH

OH

Serine

Threonine

C

H2N

CO2H

H2N

H2N

C

CO2H

LH2

OH

(b)

Tyrosine

Phosphorylation of protein 1 induces an activating conformational change due to the energetically favourable phosphorylation (P) of a hydroxyl group (OH). ATP

K Protein kinase

OH

ADP P

* Inactive signalling protein 1 Pi (c)

Phosphatase

Activated signalling protein 1

The initiation of a phosphorylation cascade. Phosphorylated protein 1 acts as a kinase and phosphorylates protein 2. Activated protein 1 P ATP

*

ADP

* OH Inactive signalling protein 2 Pi

P Phosphatase

Figure 3.5  Intracellular signalling via phosphorylation. (a) Amino acids serine, threonine and tyrosine carry polar hydroxyl (OH) groups that can be phosphorylated. Over 99% of all protein phosphorylation occurs on serine and threonine residues; however, phosphorylation of tyrosine, the only amino acid with a phenolic ring, generates particularly distinctive intracellular signalling pathways. (b) Protein 1 is inactive until its hydroxyl group is phosphorylated by the action of a kinase enzyme. This induces a conformational change, resulting in an activated phosphorylated protein. Energy for the

Activated signalling protein 2

transfer of the phosphate group comes from the hydrolysis of ATP to ADP. The reverse reaction, from active to inactive states, is catalyzed by a phosphatase and releases inorganic phosphate (Pi) for reincorporation back into ATP. (c) The initiation of a signalling cascade. Activated phosphorylated protein 1 itself acts as a kinase and catalyzes the phosphorylation of protein 2. Amino acid specificity means that serine/threonine kinases usually show no activity with tyrosine residues and tyrosine kinases normally do not phosphorylate serine or threonine residues.

Chapter 3: Molecular basis of hormone action  /  33

I Receptor structure. Disulphide bridges (S-S) link the α-subunits of the insulin receptor to one another and also to two identical β-subunits, which span the plasma membrane

α

Links to the MAPK pathway. Grb2 links β S IRS1 to the GDP/GTP exchange protein, SoS. The Grb2–Sos complex is brought into proximity of the small G-protein, Ras, located in the plasma P membrane. Translocation of P Grb2–Sos triggers Ras by the exchange of GTP for GDP. P P The serine/threonine kinase P P enzyme, Raf, activates a tyrosine/serine/threonine P IRS1 P kinase enzyme (MEK). MEK activates MAPK. MAPK is multifunctional P P and modulates cell proliferation, differentiation and function. Ras

S

S

S

S

S

S

α

Insulin binding. Insulin I binding to the α-subunits results in autophosphorylation P of the intracellular domains of the β-subunits by the intrinsic tyrosine kinase activity ( )

Activation of IRS1 or IRS2. Autophosphorylation of the intracellular domains of the receptor causes docking of IRS1 or IRS2, which can then be activated by tyrosine phosphorylation P

S β

P

P P

P P

b2

P

P P IRS1/2

Gr

P

SoS

Glycogen synthase activation

f

Ra

MAPK

P

P

P PI3 kinase Glucose transporter translocation

M

EK

PI3 kinase pathway. IRS1 or IRS2 can activate the PI3 kinase pathway, which, in particular, enhances glucose transport

Glucose uptake Figure 3.6  The insulin receptor and some of its signalling pathways. The number of insulin receptors on target cells varies, commonly from 100 to 200,000, with adipocytes and hepatocytes expressing the highest numbers. Not all insulin-signalling pathways are shown, e.g. type 2 growth factor receptor-bound

These molecular events lie at the heart of insulin’s clinical action (see Part 3). Defects in the signalling pathway can result in resistance to insulin action either as rare monogenic syndromes (Box 3.4) or as a considerable part of the type 2 diabetes phenotype (see Chapters 11 and 13).

protein (Grb2) can be stimulated independently of insulin receptor substrate 1 (IRS1). MAPK, mitogenactivated protein kinase; PI3 kinase, phosphatidylinositol-3-kinase; SoS, son of sevenless protein; I, insulin.

Receptors that recruit tyrosine kinase activity The sub-family of receptors that bind growth hormone (GH) and prolactin (PRL) includes those for numerous cytokines and the hormones leptin

34  /  Chapter 3: Molecular basis of hormone action

Box 3.4  Defects in the insulin signalling pathways and ‘insulin resistance’ syndromes Over 50 mutations have been reported in the insulin receptor (IR) that impair glucose metabolism and raise serum insulin (‘insulin resistance’). Historically, these have been discovered as different congenital syndromes; however, the advance of molecular genetics has unified these diagnoses as a phenotypic spectrum according to the severity of IR inactivation. Patients with milder insulin resistance and less affected IR signalling are usually only diagnosed at puberty, whereas what was known as ‘Leprachaunism’, with an effective absence of functional IR, manifests as severe intrauterine growth retardation. These latter patients rarely survive beyond the first year of life. Interestingly, the IR gene is seemingly normal in most patients with milder congenital insulin resistance, suggestive of abnormalities in other components of insulin signalling pathways. Indeed, some of these monogenic causes of insulin resistance have now been discovered. Impaired insulin signalling is also a very significant component of type 2 diabetes (see Chapters 11 and 13).

and erythropoietin (EPO). The basic receptor composition, shown in Figure 3.2, contains major homology between family members in the extracellular domain. Growth hormone and prolactin signalling pathways – the Janus family of tyrosine kinases Similar mechanisms govern GH and PRL receptor binding and signal transduction. Two different sites on the hormone are capable of binding receptors that dimerize. The hormone–dimerized receptor interaction leads to conformational change in the

cytoplasmic regions and signal transduction. Discovery of this phenomenon has been utilized in drug design to combat excessive GH action in acromegaly (Figure 3.7). The EPO receptor also forms homodimers, i.e. two identical receptors bind together. The cytokine receptors tend to form heterodimers with diverse partner proteins. Activation of the hormone receptor rapidly recruits one of four members of the ‘Janus-associated kinase’ (JAK) family of TKs (Figure 3.8), so named after the two-faced Roman deity, Janus, because of distinctive, tandem kinase domains at their carboxyterminals. GH, PRL and EPO receptor dimerization brings together JAK2 molecules that become phosphorylated. The major downstream substrates of JAK are the STAT family of proteins (hence the term ‘JAK-STAT’ signalling; Figure 3.8). The name STAT comes from dual function: signal transduction, located in the cytoplasm, and nuclear activation of transcription. Both activities rely on phosphorylation by JAK (Figure 3.8). Phosphorylated STAT proteins dissociate from the occupied receptor–kinase complex and, themselves, dimerize to gain access to the nucleus. There, they activate target genes, commonly those that regulate proliferation or the differentiation status of the target cell. One of the major targets of GH is the IGF-I gene (Box 3.5). JAK signalling does not focus exclusively on STAT. The GH receptor (GHR) also signals through MAPK and PI3-kinase pathways. This overlap may account for some of the rapid metabolic effects of GH (Figure 3.8). Defects in the GH signalling pathway can result in rare syndromes of resistance to GH action (Box 3.6 and Figure 3.9).

G-protein–coupled receptors The commonest subset of cell-surface receptors (>140 members) couple to G-proteins at the inner surface of the cell membrane, leading to the generation of intracellular second messengers such as adenosine-3′, 5′-cyclic monophosphate (cyclic AMP or cAMP), diacylglycerol (DAG) and inositol triphosphate (IP3). In addition to hormones, G-protein–coupled receptors (GPCRs) also exist for glutamate, thrombin, odourants and the visual transduction of light.

Chapter 3: Molecular basis of hormone action  /  35

GH Dimerized GH receptor The GH receptor antagonist, pegvisomant (PEG), inhibits GH signalling. It binds (with increased affinity) to the dimerized GH receptor, but fails to induce twisting, so preventing recruitment of JAK2.

Cell membrane

GH binds to the dimerized receptor to induce twisting of the intracellular domain and exposure of the JAK2 docking site

PEG

GH

JAK2

Dimerized GH– receptor complex recruits and activates JAK2

GH

JAK2

JAK2

Figure 3.7  Growth hormone (GH) signalling and its antagonism. GH binds to its cell-surface receptors and, via altered conformation of the receptor dimer, recruits Janus-associated kinase 2 (JAK2). This model led to the design of the GH receptor antagonist, pegvisomant.

Box 3.5  One of the major targets of GH signalling is the IGF-I gene • Measuring serum IGF-I is a useful measure of GH activity in the body

The most striking structural feature of all these receptors lies in the transmembrane domain, comprising hydrophobic helices, which cross the lipid bilayer of the plasma membrane seven times (Figure 3.10). GPCR signalling involves the hydrolysis of GTP to GDP. In their resting state, the G-proteins

exist in the cell membrane as heterotrimeric complexes with α, β and γ subunits. In practice, the β and γ subunits associate with such affinity that the functional units are Gα and Gβ/γ. Hormone occupancy results in conformational change of receptor structure. In turn, this causes a conformational change in the α-subunit, leading to an exchange of GDP for GTP. The acquisition of GTP causes the α-subunit to dissociate from the Gβ/γ subunits and bind to a downstream catalytic unit, either adenylate cyclase in the generation of cAMP or phospholipase C (PLC) to produce DAG/IP3 from phosphatidylinositol (Figures 3.10 and 3.11). The energy to activate these target enzymes comes

36  /  Chapter 3: Molecular basis of hormone action

GH binding. Binding of growth hormone to the dimerized receptor recruits the cytoplasmic tyrosine kinase, JAK2

STATs. Activated JAK2 phosphorylates tyrosines on STATs. These form dimers and undergo translocation into the nucleus, where they act as transcriptional regulatory proteins

JAK2 binding to 'Box 1'. JAK2 binds to the juxtamembrane region of the intracellular domains of the receptor, known as Box 1 ( )

GH

JAK

2

P

JAK

2

P

P

STAT P

MAPK

P

P

P

P

P

STAT

IRS1/2

P

P

P

P STAT STAT

P

P

PI3 kinase

Rapid metabolic effects

NUCLEUS: MAPK pathway. The intermediate steps leading to MAPK activation by GH are similar to those shown for insulin in Figure 3.6

Gene regulation (e.g. SRE, c-fos)

PI3 kinase pathway. The intermediate steps, whereby IRS1 and IRS2 recruit the p85 subunit of PI3 kinase, are the same as for insulin, and are responsible for some of the rapid metabolic effects of GH

Figure 3.8  Growth hormone (GH) receptor and its signalling pathways. The receptor recruits tyrosine kinase (TK) activity from Janus-associated kinase 2 (JAK2). Serum response elements (SREs) are mitogen-activated protein kinase (MAPK)-responsive

regulatory elements that mediate the induction of target genes, such as c-fos. STAT, signal transduction and activation of transcription protein; PI3 kinase, phosphatidylinositol-3-kinase; IRS, insulin receptor substrate.

from the cleavage of phosphate from GTP. This regenerates Gα-GDP, which no longer associates with adenylate cyclase or PLC, thus switching off the cascade and recycling the Gα-GDP back to the start. There are over 20 isoforms of the Gα subunit that may be grouped into four major sub-families (Box 3.7). These are involved differentially with the various hormone receptor signalling pathways (Table 3.1). More than half of GPCRs can interact with different Gα subunits and thus signal through contrasting, and sometimes opposing, intracellular

second messenger systems (Figure 3.12). In part, this promiscuity can be attributed to the degree of hormonal stimulation or activation of different receptor sub-types. For instance, at low concentrations, TSH, calcitonin and LH receptors associate with Gsα to activate adenylate cyclase, whereas higher concentrations recruit Gqα to activate PLC. Calcitonin receptor sub-types are differentially expressed according to the stage of the cell cycle. Defects in the G-protein signalling pathways can result in many endocrine disorders (Box 3.8; Figures 3.13 and 3.14).

Chapter 3: Molecular basis of hormone action  /  37

Box 3.6  Defects in growth hormone signalling pathways and growth hormone resistance syndromes Severe resistance to GH, mainly secondary to mutations in the GH receptor that commonly affect the hormone-binding domain, is characterized by grossly impaired growth and is termed Laron syndrome, eponymously named after it was first reported by Laron in 1966 (Figure 3.9). It is an autosomal recessive disorder with a variable phenotype typified by normal or raised circulating GH and low levels of serum IGF-I. For other patients, no GHR mutations have been identified, implicating genes that encode downstream components or related aspects of GH signalling. For instance, defects in the IGF-I gene have been associated with severe intrauterine growth retardation, mild mental retardation, sensorineural deafness and postnatal growth failure.

Second messenger pathways

Figure 3.9  Laron syndrome showing truncal obesity. This boy presented aged 10.4 years but with a height of 95 cm – equivalent to that of a 3-year-old. In addition to truncal obesity, there is a very small penis. These features could represent severe growth hormone (GH) deficiency. However, serum GH levels were elevated with undetectable insulin-like growth factor I (IGF-I) indicative of GH resistance. Laron syndrome was diagnosed due to an inactivating mutation of the gene encoding the GH receptor. Other clinical features include a prominent forehead, depressed nasal bridge and under-development of the mandible.

Cyclic adenosine monophosphate Activation of membrane-bound adenylate cyclase catalyzes the conversion of ATP to the potent second messenger cAMP (see Figure 3.11). cAMP interacts with protein kinase A (PKA) to unmask its catalytic site, which phosphorylates serine and threonine residues on a transcription factor called cAMP response element binding protein (CREB) (Figure 3.15). CREB then translocates to the nucleus where it binds to a short palindromic sequence in the regulatory regions of cAMPregulated genes. This signalling pathway controls major metabolic pathways, including those for lipolysis, glycogenolysis and steroidogenesis. The cAMP response is terminated by a large family of phosphodiesterases (PDEs), which can be activated by a variety of systems, including phos-

phorylation by PKA in a negative feedback loop. PDEs rapidly hydrolyze cAMP to the inactive 5′AMP. In addition, activated PKA can phosphorylate serine and threonine residues of the GPCR to cause receptor desensitization. Diacylglycerol and Ca2+ Signalling from hormones, such as TRH, GnRH and oxytocin, recruits G-protein complexes containing the Gqα subunit. This activates membraneassociated PLC, which catalyzes the conversion of PI 4,5-bisphosphate (PIP2) to DAG and IP3 (Figures 3.11 and 3.16). IP3 stimulates the transient release of calcium from the endoplasmic reticulum to activate several calcium-sensitive enzymes, including

38  /  Chapter 3: Molecular basis of hormone action

Vacant receptor

'Resting' G-protein

NH2

'Resting' adenylate cyclase

Plasma membrane

N

N N

N CH2 O

γ

β

Adenosine-3',5'-cyclic monophosphate

C

CH2.O.CO.R1

5' 4'

P

1' 3'

α

CH.O.CO.R2

2'

CH2.OH

OH

GDP

Diacylglycerol (DAG)

+ Hormone (

)

CH2.O.CO.R1

OH HO β

GTP binds

α GDP

4

γ

C

OH 3

OH

GDP released

6

2

γ

C*

OH P

CH.O.CO.R2

1

P

5 4

CH2

6

P 3

OH

Phosphatidylinositol (PI)

+ Conformational change

β

5

OH

1 2

P

OH

Inositol-1,4,5-tris phosphate (IP3)

Figure 3.11 Second messengers that mediate G-protein–coupled receptor signalling. The symbol P is the abbreviation for a phosphate group. Carbon atoms are numbered in their ring position. R1 and R2 represent fatty acid chains.

α GTP

ATP cAMP

Figure 3.10  G-protein–coupled receptors. The extracellular domain is ligand specific and, hence, less conserved across family members (e.g. only 35–45% for the TSH, LH and FSH receptors). The transmembrane domain has a characteristic heptahelical structure, most of which is embedded in the cell membrane and provides a hydrophobic core. Conserved cysteine residues can form a disulphide bridge between the second and third extracellular loops. The cytoplasmic domain links the receptor to the signal-transducing G-proteins and, in this example, is linked to membrane-bound adenylate cyclase. The activation of adenylate cyclase is depicted by the conversion of C to C*.

Box 3.7  Sub-families of Gα protein subunits • Gsα: activates adenylate cyclase • Giα: inhibits adenylate cyclase • Gqα: activates PLC • Goα: activates ion channels

isoforms of protein kinase C (PKC), and proteins like calmodulin (Figure 3.16). Calcium ions also activate cytosolic guanylate cyclase, an enzyme that catalyzes the formation of cyclic guanosine monophosphate (cGMP). The effects of atrial natriuretic peptide are mediated by receptors linked to guanylate cyclase. The major target of DAG signalling is PKC, which activates phospholipase A2 to liberate arachidonic acid from phospholipids and generate potent eicosanoids, including thromboxanes, leucotrienes,

Chapter 3: Molecular basis of hormone action  /  39

Table 3.1  Use of different G-protein α-subunits by various hormone signalling pathways Hormone

Dominant G-protein α-subunit(s)

Thyrotrophin-releasing hormone (TRH)

Gqα

Corticotrophin-releasing hormone (CRH)

Gsα

Gonadotrophin-releasing hormone (GnRH)

Gqα

Somatostatin (SS)

Giα/Gqα

Thyroid-stimulating hormone (TSH)

Gsα/Gqα

Luteinizing hormone (LH)/human chorionic gonadotrophin (hCG)

Gsα/Gqα

Follicle-stimulating hormone (FSH)

Gsα/Gqα

Adrenocorticotrophic hormone (ACTH)

Gsα

Oxytocin

Gqα

Vasopressin

Gsα/Gqα

Catecholamines (β-adrenergic)

Gsα

Angiotensin II (AII)

Giα/Gqα

Glucagon

Gsα

Calcium

Gqα/Giα

Calcitonin

Gsα/Giα/Gqα

Parathyroid hormone (PTH)/PTH-related peptide (PTHrP)

Gsα/Gqα

Prostaglandin E2

Gsα

Hormone binds to receptor

Activation of G-protein Gs / Gi / Gq / Go

Activation / inhibition of adenylate cyclase

I / I cAMP

For signalling by SS, vasopressin, AII, calcitonin and PTH/PTHrP, different receptor sub-types, potentially in different tissues, determine α-subunit specificity. This provides opportunities for selective antagonist therapies.

Activation of phospholipase C

I Ca2+, DAG, IP3

Hormone receptor

G-protein

Catalytic subunit

Second messenger

Figure 3.12  Hormonal activation of G-protein– coupled receptors can link to different second messenger pathways. The two alternative pathways are not mutually exclusive and may, in fact, interact.

Figure 3.13  Familial male precocious puberty (‘testotoxicosis’). This 2-year-old presented with signs of precocious puberty. Note the musculature, pubic hair and size of the testes and penis. He was the size of a 4-year-old. His overnight gonadotrophins [luteinizing hormone (LH) and follicle stimulating hormone] were undetectable as the testosterone was arising autonomously from Leydig cells due to a gain-of-function mutation in the gene encoding the LH receptor (see Box 3.8).

40  /  Chapter 3: Molecular basis of hormone action

Box 3.8  Defects in the G-protein– coupled receptor/G-protein signalling pathways Several endocrinopathies occur because of activating or inactivating mutations in genes encoding GPCRs or G-proteins coupled to them. Activating mutations cause constitutive overactivity; inactivating mutations cause hormone resistance syndromes characterized by high circulating hormone levels but diminished hormone action. Gain of function • LH receptor: male precocious puberty (Figure 3.13) • TSH receptor: ‘toxic’ thyroid adenomas • Gsα: McCune–Albright syndrome (Figure 3.14), some cases of acromegaly and some autonomous thyroid nodules Loss of function • V2 receptor: nephrogenic diabetes insipidus (high vasopressin) • TSH receptor: resistance to TSH (high TSH) • Gsα: pseudohypoparathyroidism (see Figure 9.9) and Albright hereditary osteodystrophy

(a)

Figure 3.14  McCune–Albright syndrome. At 6 years of age, this girl presented with breast development and vaginal bleeding in the absence of gonadotrophins. An activating mutation in Gsα had created independence from melanocyte-stimulating hormone (MSH) causing skin pigmentation (‘café-au-lait’ spots) and similar constitutive activation in the ovary (i.e. independence from gonadotrophins), giving rise to

lipoxins and prostaglandins (Figure 3.17). The latter are well-recognized paracrine and autocrine mediators capable of amplifying or prolonging a response to a hormonal stimulus.

Nuclear receptors The second superfamily of hormone receptor is the nuclear receptors, which are classified by their ligands, small lipophilic molecules that diffuse across the plasma membrane of target cells. Once ligand bound, the receptors typically bind DNA and function as transcription factors (Figure 3.18). This need for transcription and translation to elicit an effect means that biological responses of nuclear receptors are relatively slow compared to cell-surface receptor signalling. Distinct regions of nuclear receptors can be identified, for which evolutionary conservation can be as high as 60–90%, i.e. the receptors are structurally related (Figure 3.19). For one sub-group of the superfamily, no endogenous ligand has been identified and they are termed ‘orphan’ nuclear receptors. In addition, some variant receptors have atypical DNA-binding domains and potentially function via indirect interaction with the genome. All the different types are associated with endocrinopathies, usually due to loss of function. (b)

premature breast development. In some cases, constitutive overactivity can manifest in bones (causing ‘fibrous dysplasia’), the adrenal cortex (Cushing syndrome) and the thyroid (thyrotoxicosis). From Brook’s Clinical Pediatric Endocrinology, Sixth Edition, Charles G. D. Brook, Peter E. Clayton, Rosalind S. Brown, Eds. Blackwell Publishing Limited. 2009.

Chapter 3: Molecular basis of hormone action  /  41

Inactive kinase cAMP tetramer

cAMP / Regulating Active subunit complex kinase +

+

+

Catalyzes phosphorylation of CREB

RNA POL

5'

Target gene

at much higher concentrations than aldosterone, might saturate the MR, causing inappropriate overactivity. Accordingly, impaired function of HSD11B2 leads to hypertension and hypokalaemia in the syndrome of ‘apparent mineralocorticoid excess’. Nuclear localization, DNA binding and transcriptional activation

3'

CRE

Figure 3.15  The activation of protein kinase A, a cAMP-dependent protein kinase. The four-subunit complex is inactive. When cAMP binds to the regulatory subunits (red), dissociation occurs so that the active kinase subunits (blue) are released to catalyze the phosphorylation of the cAMP response element-binding protein (CREB). This activates CREB (  →  ) so that it can bind to its DNA target, the cAMP response element (CRE), to switch on transcription of cAMP-inducible genes. RNA POL, RNA polymerase.

The receptors predominantly reside in the nucleus, although increasingly nuclear import and export appears to be an important regulatory mechanism, controlling access of the nuclear receptor to target gene DNA. This shuttling has been long recognized for the glucocorticoid receptor (Figure 3.18). Target cell conversion of hormones destined for nuclear receptors In many instances, the ligand for the nuclear receptor undergoes enzymatic modification within the target cell. This converts the circulating hormone into a more or less potent metabolite prior to receptor binding (Table 3.2). For instance, cortisol is metabolized to cortisone by type 2 11β-hydro­ xysteroid dehydrogenase (HSD11B2). In kidney tubular cells, this inactivation preserves aldosterone action at the mineralocorticoid receptor (MR). Without this, cortisol, present in the circulation

In their resting state, unbound steroid hormone receptors associate with heat-shock proteins, which obscure the DNA-binding domain, so that they are considered incapable of binding the genome. Steroid binding causes conformational change and dissociation of the heat-shock proteins. This reveals two polypeptide loops stabilized by zinc ions, known as zinc fingers. Once two steroid receptors have dimerized, these motifs bind to target DNA at the specific hormone response element (HRE) (Figure 3.20). The unliganded thyroid hormone receptor (TR) is located in the nucleus bound to DNA at the thyroid hormone response element (TRE). In the absence of hormone, the TR dimerizes with the retinoid X receptor and tends to recruit nuclear proteins that inhibit transcription (co-repressors). The binding of thyroid hormone leads to dissociation of these factors, the recruitment of transcriptional co-activators, and a sequence of events recruiting DNA-dependent RNA polymerase leading to transcription (Figure 3.20 and review Figure 2.2). Resistance syndromes for nuclear receptors are similar to those for cell-surface receptors. Inactivating mutations lead to loss of receptor function, such as reduced hormone binding or impaired receptor dimerization, loss of hormone action, for instance decreased binding to the HRE, and, characteristically, raised circulating hormone levels (Table 3.3). Orphan nuclear receptors and variant nuclear receptors Some orphan and variant receptors play very important roles in endocrinology. For instance,

42  /  Chapter 3: Molecular basis of hormone action

H Cell membrane

PI

PIP

PIP2

Diacylglycerol β γ

α

Phospholipase C

+

Protein kinase C

I IP2

IP

Protein

+

IP3

P -Protein Cytosol Substrate

Ca2+ +

+

Ca2+-sensitive enzymes

Modified substrate

+

Endoplasmic reticulum +

Calmodulin

Calmodulinactivated protein kinase

Protein

P -Protein

Figure 3.16  Hormonal stimulation of intracellular phospholipid turnover and calcium metabolism. Phosphatidylinositol (PI) metabolism includes the membrane intermediaries, PI monophosphate (PIP) and PI bisphosphate (PIP2). Hormone action stimulates phospholipase C, which hydrolyzes PIP2 to diacylglycerol (DAG) and inositol triphosphate (IP3).

IP3 mobilizes calcium, particularly from the endoplasmic reticulum. DAG activates protein kinase C and increases its affinity for calcium ions, which further enhances activation. Collectively, these events stimulate phosphorylation cascades of proteins and enzymes that alter intracellular metabolism.

steroidogenic factor 1 (SF1, also called NR5A1) is a critical mediator of endocrine organ formation. Without it, the anterior pituitary gonadotrophs, adrenal gland and gonad fail to develop. It is also critical for the ongoing expression of many important genes within these cell types (e.g. the enzymes that orchestrate steroidogenesis; see Figure 2.6). A variant receptor with a similar expression profile is DAX1 (also called NROB1), mutation of which

causes congenital adrenal hypoplasia (i.e. underdevelopment). Duplication of the region that includes the gene encoding DAX1 causes maleto-female sex reversal (see Chapters 6 and 7). Increasingly, endogenous compounds are being identified that occupy the three-dimensional structure created by the ligand-binding domain. Whether these substances are the true hormone ligands remains debatable.

Chapter 3: Molecular basis of hormone action  /  43

Membrane phospholipid C

O

CH2

O

CH

O

CH2 O

P

O C O

Phospholipase A2 COOH

O

O –

Arachidonic acid

O COOH OH

PGE2

OH

Figure 3.17  Eicosanoid signalling. Arachidonic acid, released by phospholipase A2, is the rate-limiting precursor for generating eicosanoid signalling molecules by cyclo-oxygenase (COX) and lipoxygenase pathways. This example produces prostaglandin E2 (PGE2) but there are at least 16 prostaglandins – structurally related, 20-carbon, fatty acid derivatives. They are released from many cell

Endocrine transcription factors Although distinct from the nuclear receptor superfamily, other transcription factors play critical roles in the endocrine system, both during its development and in regulating its differentiated function (Table 3.4). This is important to the endocrinologist because inactivating mutations in the transcription factors can be a cause of endocrine pathology, particularly in the paediatric clinic, where molecular genetics can increasingly provide precise diagnostic answers (see Chapter 4). For instance, in the pituitary, pituitary-specific transcription factor 1 (PIT1) regulates the expression of genes encoding GH, PRL and the β-subunit of TSH. Patients with inactivating PIT1 mutations

types and exert paracrine and autocrine actions (e.g. the inflammatory response and contraction of uterine smooth muscle). Their circulating half-life is short (3–10 min). Aspirin inhibits prostaglandin production at sites of inflammation. There are different forms of COX; inhibitors of COX-2 are also used as anti-inflammatory agents.

show reduced, or absent, levels of these hormones, causing short stature, and are at risk of congenital secondary hypothyroidism with severe learning disability. The development of the pancreas and, in particular, the specification and function of β-cells relies on several transcription factors. Pancreas duodenal homeobox factor 1 [PDX1, also called insulin promoter factor 1 (IPF1)] and several members of the hepatocyte nuclear factor (HNF) family are critical in this regard; inactivating mutations have been identified, which cause monogenic diabetes mellitus at an early age, called maturity-onset diabetes of the young (MODY) (see Table 11.3). Potentially, these patients never accrue a normal number of β-cells, which also fail to function properly.

44  /  Chapter 3: Molecular basis of hormone action

Serum

Bound steroid hormone

Free steroid hormone

Serum binding protein

(a)

Cell membrane Diffusion

(g)

(b)

Post-translational modification

Protein

Cytosol

R

R

R*

R

Ribosome–mRNA complex

(f)

(c)

(e) Nucleus

R* (d)

Mature mRNA

Chromatin DNA-dependent RNA polymerase

Figure 3.18  Simplified schematic of nuclear hormone action. (a) Free hormone (a steroid is shown), in equilibrium with that bound to protein, diffuses across the target cell membrane. (b) Inside the cell, free hormone binds to its receptor ( R ). This may occur in the cell cytoplasm (e.g. glucocorticoid receptor) or in the cell nucleus (e.g. thyroid hormone receptor). (c) The activated hormone–receptor complex ( R *), now

Transcription

present in the nucleus, binds to the hormoneresponse element of its target genes. (d) This interaction promotes DNA-dependent RNA polymerase (Pol II) to start transcription of mRNA. (e) Post-transcriptional modification and splicing sees the mRNA exit the nucleus for translation into protein on ribosomes. Post-translational modification provides the final protein.

Chapter 3: Molecular basis of hormone action  /  45

N

Mineralocorticoid

N

Progesterone Androgen

N N

Glucocorticoid N

Oestrogen Calcitriol

N N N

Retinoic acid Tri-iodothyronine

Highly conserved, DNA-binding domain, comprised of two zinc fingers Specific hormone-binding domain, which forms a hydrophobic pocket C-terminus ‘AF2’ region, which recruits the nuclear components for transcriptional activation Variable N-terminal domain Figure 3.19  The nuclear hormone receptor superfamily. The receptors, named according to their ligands (shown to the right), range in size from 395 to 984 amino acids.

Table 3.2  Potential modifications of hormones, their precursors and metabolites within the cell prior to nuclear receptor action Modification that increases activity

Modification that decreases activity

Deiodination of thyroxine (T4) to tri-iodothyronine (T3) by type 1 and type 2 selenodeiodinase (see Figure 8.6)

Inactivation of T4 and T3 by the formation of reverse T3 and di-iodothyronine (T2) by type 3 selenodeiodinase (see Figure 8.6)

Reduction of testosterone to dihydrotestosterone (DHT) by 5α-reductase (see Figure 7.7); gain of oestrogenic activity by conversion of testosterone to oestradiol by the action of aromatase (CYP19; see Figure 2.6)

Loss of androgenic activity by conversion of testosterone to oestradiol by the action of aromatase (CYP19; see Figure 2.6)

Conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D (calcitriol) by 1αhydroxylase (see Figure 9.2)

Conversion of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D or the inactivation of 1,25-dihydroxyvitamin D to 1,24,25-trihydroxyvitamin D by 24α-hydroxylase (see Figure 9.2)

Generation of cortisol from cortisone by type 1 11β-hydroxysteroid dehydrogenase (HSD11B1; see Figure 6.4)

Inactivation of cortisol to cortisone by Type 2 11β-hydroxysteroid dehydrogenase (HSD11B2; see Figure 6.4)

The biological importance of these modifying enzymes is exemplified by rare mutations in the genes that encode them, presenting with endocrine overactivity or underactivity.

46  /  Chapter 3: Molecular basis of hormone action

(a) Steroid receptors form homodimers

(b) The thyroid hormone receptor forms heterodimers

RXR

TR

DNA = = = HRE =

HRE Steroid bound to hormone binding domain Zinc fingers Hexanucleotide half-sites arranged palindromically Hormone-response element

Figure 3.20  Nuclear hormone receptor–DNA interactions. (a) Steroid hormone receptors form homodimers bound to palindromic hexanucleotide target DNA sequences that comprise the hormone response element (HRE). (b) Thyroid hormone receptor (TR), similar to receptors for retinoic acid and calcitriol, forms heterodimers with the retinoid X receptor. (c) Once occupied by tri-iodothyronine (T3), DNAbound TR recruits co-activator proteins which, in turn, bridge to, activate and stabilize the multiple components of the transcription initiation complex at the basal promoter of the target gene.

DNA TRE = TR = = RXR =

T3 Thyroid hormone receptor Zinc fingers Retinoid X receptor, which is forming a heterodimer with the TR = Direct repeat configuration of half-sites TRE = Thyroid hormone-response element

(c) Co-activator RXR

TR

TIC RNA POL Target gene DNA

TRE = T3 bound to the hormone binding domain of the thyroid hormone receptor (TR) = Zinc fingers RXR = Retinoid X receptor TIC = Transcription initiation complex RNA POL = RNA polymerase TRE = Thyroid hormone-response element

Table 3.3  Defects in nuclear hormone signalling Mutations in receptor for

Clinical effects

Androgen (AR)

Partial or complete androgen insensitivity syndromes

Glucocorticoid (GR)

Generalized inherited glucocorticoid resistance

Oestrogen (ER)

Oestrogen resistance

Thyroid hormone (TR)

Thyroid hormone resistance

Vitamin D (VDR)

Vitamin D (calcitriol)-resistant rickets

Chapter 3: Molecular basis of hormone action  /  47

Table 3.4  Some important transcription factors required for development and function of endocrine cell types and organs Organ or cell type

Transcription factor

Adrenal gland

SF-1, DAX1, CITED2

Enteroendocrine cells

NGN3

Gonad

WT1, SRY, SOX9, SF-1, DAX1

Pancreas/islets of Langerhans

PDX1, SOX9, HLXB9, NGN3, PAX6, PAX4, RFX6, NKX2.2, NKX6.1, NeuroD1 (also see Table 13.3)

Parathyroid gland

TBX1 (part of Di George syndrome; see Figure 4.4), GATA3

Pituitary

PIT1, PROP1, HESX1, PITX2, SF-1, DAX1, LHX3, LHX4

Thyroid gland

PAX8, FOXE1, NKX2.1

Key points • Hormones act by binding to receptors and triggering intracellular responses • Tissue distribution of the receptor determines where a hormone will exert its effect • The two major subdivisions of hormone receptors are classified by site of action: cell surface and nuclear • Peptide hormones and catecholamines act via cell-surface receptors and generate fast responses in seconds or minutes • Steroid and thyroid hormones act via nuclear receptors to alter expression of target genes; a slow response occurs because of the need to produce protein from the expression of target genes • Mutations in genes encoding any part of the cascade from hormone receptor to action can result in underactive or overactive endocrinopathy, or potentially tumour formation

48

CHAPTER 4

Investigations in endocrinology and diabetes Key topics ■ Laboratory assay platforms

49

■ Cell and molecular biology as diagnostic tools

56

■ Imaging in endocrinology

57

■ Key points

61

Learning objectives ■ To understand how circulating hormones are measured by a

range of different immunoassays ■ To understand other laboratory investigations as applied to

clinical endocrinology and diabetes ■ To understand the molecular biology that underpins genetic

diagnoses ■ To understand the options available for imaging the

endocrine system This chapter details how clinical endocrinology and diabetes is investigated

Essential Endocrinology and Diabetes, Sixth Edition. Richard IG Holt, Neil A Hanley. © 2012 Richard IG Holt and Neil A Hanley. Publlished 2012 by Blackwell Publishing Ltd.

Chapter 4: Investigations in endocrinology and diabetes  /  49

All specialties have been advanced by methods to aid diagnosis, and monitor and assess treat­ ment. Investigation in endocrinology and diabetes remains centred on laboratory assays that determine the concentration of hormones and metabolites usually in blood. The first challenge is correct col­ lection; for some investigations, prior fasting is important. Appropriate conditions or preservatives are mandatory (Box 4.1). In addition to clinical biochemistry (also called chemical pathology), molecular genetics and cytogenetics are routine investigations to provide personalized genetic diag­ noses that predict the course of some endocrine disorders (e.g. multiple endocrine neoplasia; see Chapter 10). Outside of the laboratory, clinical investigation draws heavily upon expertise in radiology and nuclear medicine. Some investigations are highly specific (e.g. visual fields for pituitary tumours or retinal screening for diabetes) and these are covered in later topic-specific chapters.

Laboratory assay platforms Immunoassays Hormones (and other metabolites) are most com­ monly measured by immunoassay, although increas­ ingly mass spectrometry is used (see below). Immunoassays, introduced in the 1960s, are suffi­ ciently sensitive, precise and hormone specific for routine application in clinical biochemistry. Bioassays, which measure physiological responses induced by a stimulus, are near obsolete in clinical practice. Immunoassay is a broad term for one of two different techniques: true immunoassay and immu­ nometric assay. Both forms are based on the hormone to be measured being antigenic and bound by specific antibodies to form an antibody–antigen complex. Both forms of immunoassay also employ a label, historically a radioactive isotope [e.g. iodine125 (I125)], but commonly now a fluorescent tracer, to generate a quantitative signal. Both assays also rely on comparison of the patient sample with known concentrations of a reference compound.

Box 4.1  The specifics of sample collection are mandatory Containers for investigation of blood • Lithium heparin: most hormones • Fluoride oxalate: glucose • Di-potassium EDTA: tests requiring DNA isolation Peptide hormones and catecholamines tend to be less stable than other hormones and need prompt delivery to the laboratory on ice Samples for which prior fasting may be required • Glucose • Lipids • Calcium Hormones that may require 9 am collection • Cortisol • Testosterone Containers for investigation of urine • Acid: calcium, 5-hydroxyindoleacetic acid (5-HIAA), catecholamines • No acid/simple preservative: urinary free cortisol

To set up a calibration or standard curve for the immunoassay, a constant amount of antibody is added to a series of tubes with increasing, known amounts of a reference preparation, in this example GH (Figure 4.1). This reaction is reversible with the antigen and antibody continuously associating and dissociating; however, after incubation, equilibrium is reached when tubes with more GH generate more bound complex. Measurement of the amount of bound complex (e.g. in terms of fluorescence or radioactivity) can thus be related to the quantity of GH that was originally added. This allows a calibra­ tion curve to be plotted, against which the same process can now deduce the GH concentration in a patient sample.

50  /  Chapter 4: Investigations in endocrinology and diabetes

Zero GH

+2 GH

+4 GH

Zero GH

Anti-GH

+4 GH

GH

Labelled second anti-GH

Incubate

Incubate Add excess labelled second anti-GH

+ excess

+ excess

Separate antibody-bound complex

GH

Figure 4.1  The basics of immunoassay are shown for growth hormone (GH; see text for details). For clarity, in Figures 4.1–4.3 only small numbers of hormone molecules and antibodies are shown; in practice, numbers are in the order of 108–1013.

Immunometric assays – the sandwich assays In the immunometric assay (shown for GH in Figure 4.2), a constant amount of antibody is added to each tube with increasing, known amounts of reference preparation. After incubation, the amount of GH bound to the antibody is detected by adding an excess of a second labelled antibody to all tubes. The second antibody is directed against a different antigenic site on GH from the first antibody to form a triple complex sandwiching GH between the two antibodies. Any unbound antibody is removed, leaving the amount of triple complex to be deter­ mined by quantifying the bound label (e.g. fluores­ cence or radioactivity). This emission is plotted for increasing, known amounts of reference compound to generate a calibration curve (Figure 4.2). In prac­ tice, five to eight concentrations of hormone standard are used to generate a precise calibration curve, against which patient samples can be inter­

Tube 1

Tube 2

Count bound fraction

Antibody-bound counts

Anti-GH

Calibration curve Counts from sample tube

Tube 2

hGH in sample tube Standard GH

Tube 1

Figure 4.2  The basics of an immunometric assay for growth hormone (GH; also see text). As in Figure 4.1, in practice large numbers of molecules are present for each reagent and the incubation of the first and second antibodies is usually simultaneous. Because the hormone is bound between the two antibodies in the triple complex ( ), this assay is sometimes referred to as a sandwich assay. Separation of the complex from the excess labelled second antibody is usually achieved physically, e.g. by precipitation and centrifugation (the supernatant contains the unbound antibody and is discarded). This leaves the bound labelled second antibody to be quantified by counting radioactivity or measuring fluorescence. The low measurement from the ‘zero’ tube, Tube 1, and the higher value from Tube 2 are plotted on the calibration curve. Tube 1 is not zero because of minor non-specific antibody binding. The calibration line is also curved, rather than straight, because of the reversible nature of the interaction between antibodies and their antigens. In practice, five to eight calibration points are used to construct the curve.

Chapter 4: Investigations in endocrinology and diabetes  /  51

polated. The immunometric assay is suitable only when the hormone to be measured permits discrete binding of two antibodies. This would not work for small hormones such as thyroxine (T4) or triiodothyronine (T3), for which the competitivebinding immunoassay system must be used.

Zero tube

Unlabelled T4 standard

+12 +12

+12

Labelled T4

Anti-T4

+T4

Incubate

Immunoassays – the competitive-binding assays

Analytical methods linked to mass spectrometry In some situations, immunoassays are unreliable or unavailable, commonly because antibodies lack sufficient specificity, or there are difficulties with measurements at low concentrations (e.g. serum testosterone in women). This leads to differences in measurements across different assay platforms that inhibit the development of internationally agreed standards for diagnosis and care. For some steroid or peptide hormones, or metabolic intermediaries, mass spectrometry (MS) is becoming increasing

+9 +9

6

Separate antibody-bound complex

3 3

6

Tube 1

Tube 2

Count bound fraction

Antibody-bound counts

In the competitive-binding immunoassay (shown for T4 in Figure 4.3), constant amounts of antibody and labelled antigen are added to each tube. A ‘zero’ tube is set up that contains labelled T4, as well as a tube that also includes a known amount of unla­ belled standard T4. Incubation allows the antigen– antibody complex to form. Since the zero tube contains twice as much labelled T4 as antibody, half of the labelled hormone will be bound and the other half will remain free (i.e. in excess). In the other tube, unlabelled and labelled T4 compete for the limited opportunity to bind antibody. The total antibody-bound T4 is separated (e.g. by precipita­ tion) and the label measured (e.g. by fluorescence or radioactivity). There will be less signal from the second tube because of competition from the unla­ belled T4; the decrease will be a function of the amount of unlabelled T4 added, i.e. the signal decreases as the amount of unlabelled T4 increases, allowing the construction of a calibration curve (Figure 4.3). For clinical use, standard T4 is replaced by the patient sample, with all other assay condi­ tions kept the same. As for immunometric assays, a five to eight point calibration curve offers sufficient precision for patient samples to be interpolated.

Calibration curve Tube 1

Counts from sample tube

Tube 2 T4 in sample tube Standard T4

Figure 4.3  The basics of an immunoassay for thyroxine (T4; also see text). As in Figure 4.1, in practice large numbers of molecules are present for each reagent. Under the conditions shown, the competition between equal amounts of labelled and unlabelled T4 in Tube 2 will be such that, on average, 50% of the antibody binding sites will be occupied by labelled T4. Because of competition between labelled and unlabelled hormone for a limited amount of antibody, this type of immunoassay is sometimes called a ‘competitive-binding’ assay. After removing unbound label (as in Figure 4.2 legend), the fluorescent or radioactive bound fraction is quantified and a calibration curve constructed. In practice, five to eight calibration points are used to construct the curve.

52  /  Chapter 4: Investigations in endocrinology and diabetes

helpful. It is applied either by itself or, for increased ability to resolve and measure substances, in tandem (MS/MS) or downstream of liquid chromatography (LC/MS) or gas chromatography (GC/MS). These approaches provide definitive identification of the relevant hormone or compound according to its chemical and physical characteristics, e.g. par­ ticularly useful for the unequivocal detection of performance-enhancing agents in sport. GC allows separation of vaporized molecules according to their chemical structure. For a sample loaded on a GC column, different components exit the column and pass to the mass spectrometer at different times. MS ionizes compounds to charge them, after which the spectrometer measures mass and charge during passage through an electromag­ netic field. This gives a characteristic mass-to-charge ratio for any one substance. As with immunoassays, patient samples can be judged against the perform­ ance of precisely known standards. LC/MS is similar to GC/MS; however, the initial separation is per­ formed in the liquid rather than the gaseous phase. Enzymatic assays Some metabolites are assayed enzymatically, fre­ quently using dye substrates that are catalyzed to products that are coloured or fluoresce. By incorpo­ rating known standards, the amount of colour or fluorescence can be used for precise quantification. For example, glycated haemoglobin (HbA1c), a measure of long-term diabetes control (see Chapter 11) can be measured in an enzymatic assay as well as by immunoassay and chromatography/MS approaches. Serum glucose can be measured by oxi­ dation to generate a product that interacts with a dye to generate colour or fluorescence in an enzy­ matic assay. Reference ranges Typical adult reference ranges are listed for a number of hormones in Table 4.1. Whenever possible, hor­ mones are measured in molar units (e.g. pmol/L) or mass units (e.g. ng/L). However, this is not possible for complex hormones such as the glycoproteins thyroidstimulating hormone (TSH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH),

because they circulate in a variety of slightly different forms (‘microheterogeneity’). In this scenario, inter­ national reference preparations are agreed, with potency expressed in ‘units’ (U) and their subdivisions [e.g. milliunits (mU)]. Potency is assigned after large collaborative trials involving many laboratories world­ wide using a range of assay platforms and physical analytical techniques. Patient results are then expressed relative to the reference data. Static and dynamic testing Most of endocrinology testing is ‘static’; the meas­ urement of hormones and metabolites as they circulate at any one time. However, rhythmical, pulsatile or variable hormone secretion makes inter­ pretation of single random samples meaningless or misleading (see Chapter 1). For some hormones, such as GH, a clinical impression can be gained from a series of six to eight measurements during the course of a day. Alternatively, dynamic testing can be necessary where, based on understanding normal physiology, responses are measured follow­ ing a stimulus. This might be metabolic, such as insulin-induced hypoglycaemia to study the expected rise in serum GH and cortisol (see Chapter 5), or the administration of glucose during a glucose tolerance test to diagnose diabetes (see Chapter 11). Alternatively, the stimulus might be hormonal, such as injecting adrenocorticotrophic hormone (ACTH; the anterior pituitary hormone) to measure secre­ tion of cortisol (the adrenocortical hormone). In this sense, fasting measurements, as required for serum lipids or commonly for glucose, could be viewed as dynamic, where fasting is the stimulus. Dynamic tests can be split into two categories: provocative ones to interrogate suspected inade­ quate function; or suppression tests, taking advan­ tage of negative feedback to investigate potential overactivity (Box 4.2). For instance, ACTH is injected to see if cortisol secretion rises in suspected adrenocortical inadequacy (Addison disease; see Chapter 6); whereas dexamethasone, a potent syn­ thetic glucocorticoid, is given to see if pituitary ACTH and consequently cortisol secretion is appropriately diminished. If it is not, it implies that the adrenal cortex is overactive (Cushing syndrome; see Chapter 6).

Chapter 4: Investigations in endocrinology and diabetes  /  53

Table 4.1  Endocrine reference ranges Adult reference hormone

Range

Units

Range

Unit

17-hydroxyprogesterone (male)

0.18–9.1

nmol/L

5.9–300

ng/dL

17-hydroxyprogesterone (female)

0.6–3.0

nmol/L

20–99

ng/dL

Adrenocorticotrophic hormone (ACTH, 9 am)

0–8.8

pmol/L

0–40

ng/L

Aldosterone (am; out of bed for 2 h; seated 5–15 min)a

100–500

pmol/L

3.6–18.1

ng/dL

Androstenedione (adult male and female)

2.1–9.4

nmol/L

60–270

ng/dL

Anti-Müllerian hormone (to indicate poor ovarian reserve)b

>7

pmol/L

>1

ng/mL

Chromogranin A (fasting)

0–5.2

nmol/L

0–250

ng/ml

140–700

nmol/L

5–25

µg/dL

Cortisol (midnight)

80–350

nmol/L

2.9–12.5

µg/dL

Cortisol (post low dose dexamethsaone)

30

U/L

–

–

Gastrin (fasting)

0–40

pmol/L

0–154

pg/mL

Glucagon (fasting)

0–50

pmol/L

0–139

pg/mL

  Fasting (normal)